Transgenic mice containing glucagon receptor gene disruptions

ABSTRACT

The present invention relates to transgenic animals, as well as compositions and methods relating to the characterization of gene function. Specifically, the present invention provides transgenic mice comprising mutations in a glucagon receptor gene. Such transgenic mice are useful as models for disease and for identifying agents that modulate gene expression and gene function, and as potential treatments for various disease states and disease conditions. The present invention also relates to diabetes and diabetic condition, as it demonstrates the role of the glucagon receptor in diabetes and diabetic conditions. The present invention further relates to weight gain and weight related conditions, such as obesity, and demonstrates the role of the glucagon receptor in weight gain and weight related conditions, such as obesity. In accordance with these aspects, the present invention provides methods and compositions useful in identifying, testing, and providing treatments for diabetes and diabetic conditions, weight gain and weight related conditions such as obesity.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/251,804, filed Dec. 6, 2000; U.S. Provisional Application No.60/266,044, filed Feb. 1, 2001 and U.S. Provisional Application No.60/271,121 filed Feb. 23, 2001, the entire contents of each areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to transgenic animals, compositions andmethods relating to the characterization of gene function.

BACKGROUND OF THE INVENTION

Diabetes is defined as a state in which carbohydrate and lipidmetabolism are improperly regulated by the hormone insulin (For review,see, e.g., Saltiel, Cell 104:517-529(2000)). Two major forms of diabeteshave been identified, type I and II. Type I diabetes represents theminor form of the disease, affecting 5-10% of diabetic patients. It isthought to result from the autoimmune destruction of theinsulin-producing beta cells of the pancreatic Islet of Langerhans.Exogenous administration of insulin typically alleviates thepathophysiology. Type II diabetes is the most common form of the diseaseand is possibly caused by a combination of defects in the mechanisms ofinsulin secretion and action. Both forms, type I and type II, havesimilar complications, but distinct pathophysiology.

Glucose is necessary to ensure proper function and survival of allorgans. While hypoglycemia produces cell death, chronic hyperglycemiacan also result in organ damage. Following a meal, the level of glucosein the blood is elevated. The balance between the utilization andproduction of glucose is maintained at equilibrium by two opposinghormones, insulin and glucagon. In response to elevated plasma levels ofglucose, pancreatic beta cells secrete insulin. Insulin, in turn, actson muscle, liver and adipose tissues to stimulate glucose uptake intothose cells. When plasma levels of glucose decrease, the pancreaticalpha cells secrete glucagon, which in turn stimulates glycolysis in theliver and release of glucose into the bloodstream.

The first stage of type II diabetes is characterized by the failure ofmuscle and/or other organs to respond to normal circulatingconcentrations of insulin. This is commonly associated with obesity, asedentary lifestyle, as well as a genetic predisposition. This isfollowed by an increase in insulin secretion from the pancreatic betacells, a condition called hyperinsulinemia. Ultimately, the beta cellscan no longer compensate, leading to impaired glucose tolerance, chronichyperglycemia, and tissue damage.

The action of glucagon on glycolysis in the liver is mediated by theglucagon receptor. It has been reported that the hyperglycemic state ofdiabetes mellitus is not only due to glucose under-utilization as aresult of decreased insulin, but also to the overproduction of glucosedue to elevated concentrations of glucagon (See, e.g., Unger, Diabetes25:136-151 (1976); Unger and Orci, Lancet 1:14-16 (1975)). The murineglucagon receptor cDNA, gene and promoter region was cloned, sequenced,and its tissue distribution studied (See, e.g., Burcelin et al., Gene164(2):305 (1995)). The 1944 base pair MRNA cds has been deposited inGenBank (Accession No.: L38613; GI No.: 603463). The murine glucagonreceptor was found to contain 13 exons located in a region of 4.0 kb.Moreover, this gene encodes a 485-amino-acid protein consisting of sevenputative transmembrane domains. The murine glucagon receptor was foundto be expressed predominantly in the liver, kidney, adrenal gland, lungand stomach. Lower levels of expression are detected in brown and whiteadipose tissue, cerebellum, duodenum and heart. A 1000-bp region of theglucagon receptor promoter has been sequenced. This region containsconsensus sequences for putative DNA-binding proteins involved in tissuespecificity (c/EBP; HNFI) or hormonal regulation (steroid receptor).Other consensus sequences are those known to function in controllingbasal promoter activity, such as AP1 and AP2.

Upon the binding of the glucagon receptor with glucagon, a signal to thecell is transduced, thereby triggering glycogen hydrolysis and glucosesynthesis. It has been reported that cyclic adenosine monophosphate(cAMP) mediates the effects of glucagon. The binding of glucagon to itscellular receptor activates adenylate cyclase to produce cAMP, raisingthe levels of intracellular cAMP. Elevation of intracellular levels ofcAMP is believed to result in glycogenolysis and gluconeogenesis, withthe resultant rise in glucose production by the liver (See, e.g., Unsonet al., Peptides 10:1171-1177 (1989)). Other cellular pathways have alsobeen suggested for the stimulation of glycogenolysis andgluconeogenesis. It has also been reported that glucagon binds toreceptors in the hepatocyte membrane that are coupled via a G-protein tophospholipase C. This protein causes the breakdown ofphosphatidylinositol 4,5 biphosphate to produce the second messengersinositol triphospate and 1,2 diacylglycerol upon the interaction ofglucagon and its receptor (See, e.g., Wakelam et al., Nature 323:68-71(1986); Unson et al., Peptides 10:1171-1177 (1989); Pittner and Fain,Biochem. J. 277:371-378 (1991)).

Obesity is a disease that affects at least 39 million Americans: morethan one-quarter of all adults and about one in five children. Eachyear, obesity causes at least 300,000 excess deaths in the U.S. andcosts the country more than $100 billion. Over the last 10 years, theproportion of the U.S. population that is obese has increased from 25percent to 32 percent. Obesity is measured by Body Mass Index, or BMI,which is a mathematical calculation used to determine if a person isobese or overweight. BMI is calculated by dividing a person's bodyweight in kilograms by their height in meters squared. A BMI of 30 orgreater is considered obese, while a BMI of 25-29.9 is consideredoverweight. However, the criteria for diagnosis can be misleading forpeople with more muscle mass and less body fat than normal, such asathletes. Over 70 million Americans are considered overweight. Healthproblems, including but not limited to cardiovascular disease, bloodpressure, Type II diabetes, high cholesterol, gout, certain types ofcancer, and osteoarthritis, are associated with overweight conditionsand obesity.

Diabetes and diabetic conditions, as well as weight related conditions,such as obesity, are clearly associated with health problems, and theincrease in prevalence of these conditions is a cause for concern. Aclear need exists for further analysis and, in particular, in vivocharacterization of genes, such as the glucagon receptor, to determinetheir role in dysfunctions and diseases, such as diabetes or obesity,which may play a role in preventing, ameliorating, or correctingdysfunctions or diseases.

SUMMARY OF THE INVENTION

The present invention generally relates to transgenic animals, as wellas to compositions and methods relating to the characterization of genefunction. In particular, the present invention relates to a geneencoding the glucagon receptor and its role and function in variousbiological processes and conditions, including diabetes and obesity.

The present invention provides transgenic cells comprising a disruptionin a glucagon receptor gene. The transgenic cells of the presentinvention are comprised of any cells capable of undergoing homologousrecombination. Preferably, the cells of the present invention are stemcells and more preferably, embryonic stem (ES) cells, and mostpreferably, murine ES cells. According to one embodiment, the transgeniccells are produced by introducing a targeting construct into a stem cellto produce a homologous recombinant, resulting in a mutation of theglucagon receptor gene. In another embodiment, the transgenic cells arederived from the transgenic animals described below. The cells derivedfrom the transgenic animals includes cells that are isolated or presentin a tissue or organ, and any cell lines or any progeny thereof.

The present invention also provides a targeting construct and methods ofproducing the targeting construct that when introduced into stem cellsproduces a homologous recombinant. In one embodiment, the targetingconstruct of the present invention comprises first and secondpolynucleotide sequences that are homologous to the glucagon receptorgene. The targeting construct may also comprise a polynucleotidesequence that encodes a selectable marker that is preferably positionedbetween the two different homologous polynucleotide sequences in theconstruct. The targeting construct may also comprise other regulatoryelements that can enhance homologous recombination.

The present invention further provides non-human transgenic animals andmethods of producing such non-human transgenic animals comprising adisruption in a glucagon receptor gene. The transgenic animals of thepresent invention include transgenic animals that are heterozygous andhomozygous for a null mutation in the glucagon receptor gene. In oneaspect, the transgenic animals of the present invention are defective inthe function of the glucagon receptor gene. In another aspect, thetransgenic animals of the present invention comprise a phenotypeassociated with having a mutation in a glucagon receptor gene.

In one aspect, the transgenic animals of the present invention exhibitabnormalities of the pancreas, in particular, abnormalities of thepancreatic islet cells. In a preferred aspect, the pancreas exhibits atleast one of the following characteristics: hyperplasia, hypertrophy,increased cytoplasmic vacuolization, adenomas, and granularity of thepancreatic islet cells. In a most preferred aspect, the adenomas areexhibited in aged animals, and the increased cytoplasmic vacuolizationand granularity of the pancreatic cells are exhibited in the adultanimals and the aged animals.

In another aspect, the transgenic animals of the present inventionexhibit anti-obesity characteristics. In one embodiment of this aspect,the anti-obesity characteristics include one or more of the following:reduced body weight, reduced organ weight, reduced fat as a percentageof body soft tissue, or reduced body weight gain. In a preferredembodiment of this aspect, the reduced body weight gain is seen when thetransgenic animals are exposed to a high-fat diet.

In yet another aspect, the transgenic animals exhibit abnormal bodyshape. In a preferred embodiment, the transgenic animals exhibitdwarfism.

In yet another aspect, the transgenic animals exhibit infertility. In apreferred aspect, infertility is exhibited in female transgenic animals.

In yet another aspect, the transgenic animals of the present inventionexhibit anti-diabetes-like characteristics. In one embodiment of thisaspect, the anti-diabetes-like characteristics include one or more ofthe following: decreased serum glucose levels, increased tolerance to aglucose challenge, decreased serum insulin levels, reduced body weightgain, or a decrease in fat as a percentage of body soft tissue.Preferably, the increased tolerance to a glucose challenge is observedbefore or after exposure to a high-fat diet. In accordance with thisaspect, the present invention provides transgenic animals and methodsuseful for identifying agents that ameliorate symptoms andcharacteristics of diabetes or similar diseases. In a preferredembodiment, the agent antagonizes or inhibits the activity or functionof the glucagon receptor.

The present invention also provides methods of identifying agentscapable of affecting a phenotype of a transgenic animal. For example, aputative agent is administered to the transgenic animal and a responseof the transgenic animal to the putative agent is measured and comparedto the response of a “normal” or wild-type mouse or, alternatively,compared to a transgenic animal control (without agent administration).The invention further provides agents identified according to suchmethods. The present invention also provides methods of identifyingagents useful as therapeutic agents for treating conditions associatedwith a disruption or other mutation (including naturally occurringmutations) of the glucagon receptor gene.

The present invention further provides a method of identifying agentshaving an effect on glucagon receptor expression or function. The methodincludes administering an effective amount of the agent to a transgenicanimal, preferably a mouse. The method includes measuring a response ofthe transgenic animal, for example, to the agent, and comparing theresponse of the transgenic animal to a control animal, which may be, forexample, a wild-type animal or, alternatively, a transgenic animalcontrol. Compounds that may have an effect on glucagon receptorexpression or function may also be screened against cells in cell-basedassays, for example, to identify such compounds.

The invention also provides cell lines comprising nucleic acid sequencesof a glucagon receptor gene. Such cell lines may be capable ofexpressing such sequences by virtue of operable linkage to a promoterfunctional in the cell line. Preferably, expression of the glucagonreceptor gene sequence is under the control of an inducible promoter.Also provided are methods of identifying agents that interact with theglucagon receptor gene, comprising the steps of contacting the glucagonreceptor gene with an agent and detecting an agent/glucagon receptorgene complex. Such complexes can be detected by, for example, measuringexpression of an operably linked detectable marker.

The invention further provides methods of treating diseases orconditions associated with a disruption in a glucagon receptor gene, andmore particularly, to a disruption in the expression or function of theglucagon receptor gene. In a preferred embodiment, methods of thepresent invention involve treating diseases or conditions associatedwith a disruption in the glucagon receptor gene's expression orfunction, including administering to a subject in need, a therapeuticagent that affects glucagon receptor expression or function. Inaccordance with this embodiment, the method comprises administration ofa therapeutically effective amount of a natural, synthetic,semi-synthetic, or recombinant glucagon receptor gene, glucagon receptorgene products or fragments thereof as well as natural, synthetic,semi-synthetic or recombinant analogs.

The present invention also provides compositions comprising or derivedfrom ligands or other molecules or compounds that bind to or interactwith a glucagon receptor, including agonists or antagonists of aglucagon receptor. Such agonists or antagonists of a glucagon receptorinclude antibodies and antibody mimetics, as well as other moleculesthat can readily be identified by routine assays and experiments wellknown in the art.

The present invention further provides methods of treating diseases orconditions associated with disrupted targeted gene expression orfunction, wherein the methods comprise detecting and replacing throughgene therapy mutated or otherwise defective or abnormal glucagonreceptor genes.

The present invention demonstrates the role and function of the glucagonreceptor in diabetes and diabetic conditions. The present invention alsodemonstrates the role of the glucagon receptor in weight gain and weightrelated conditions, such as obesity. In accordance with these aspects,the present invention provides methods and compositions useful inidentifying, testing, and providing treatments for diabetes and diabeticconditions, weight gain and weight related conditions such as obesity.Such methods and compositions include the human glucagon receptor geneas shown in SEQ ID NO:5 (also shown and identified in GenBank GI number:4503946; Accession number: NM_(—)000160) and any degenerates thereof,and the human glucagon receptor protein as shown in SEQ ID NO:6 (alsoshown and identified in GenBank GI number: 4503947; Accession number:NP_(—)000151) or to any deriviatives, variants, or active fragments ofthese sequences.

Definitions

The term “gene” refers to (a) a gene containing at least one of the DNAsequences disclosed herein; (b) any DNA sequence that encodes the aminoacid sequence encoded by the DNA sequences disclosed herein; and/or (c)any DNA sequence that hybridizes to the complement of the codingsequences disclosed herein. Preferably, the term includes coding as wellas noncoding regions and, preferably, includes all sequences necessaryfor normal gene expression including promoters, enhancers and otherregulatory sequences.

The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes single-, double-stranded andtriple helical molecules. “Oligonucleotide” refers to polynucleotides ofbetween 5 and about 100 nucleotides of single- or double-stranded DNA.Oligonucleotides are also known as oligomers or oligos and may beisolated from genes, or chemically synthesized by methods known in theart. A “primer” refers to an oligonucleotide, usually single-stranded,that provides a 3′-hydroxyl end for the initiation of enzyme-mediatednucleic acid synthesis. The following are non-limiting embodiments ofpolynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA,rRNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes and primers. A nucleicacid molecule may also comprise modified nucleic acid molecules, such asmethylated nucleic acid molecules and nucleic acid molecule analogs.Analogs of purines and pyrimidines are known in the art, and include,but are not limited to, aziridinycytosine, 4-acetylcytosine,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine.The use of uracil as a substitute for thymine in a deoxyribonucleic acidis also considered an analogous form of pyrimidine.

A “fragment” of a polynucleotide is a polynucleotide comprised of atleast 9 contiguous nucleotides, preferably at least 15 contiguousnucleotides and more preferably at least 45 nucleotides, of coding ornon-coding sequences.

The term “gene targeting” refers to a type of homologous recombinationthat occurs when a fragment of genomic DNA is introduced into amammalian cell and that fragment locates and recombines with endogenoushomologous sequences.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules or chromatids at the site ofhomologous nucleotide sequences.

The term “homologous” as used herein denotes a characteristic of a DNAsequence having at least about 70 percent sequence identity as comparedto a reference sequence, typically at least about 85 percent sequenceidentity, preferably at least about 95 percent sequence identity, andmore preferably about 98 percent sequence identity, and most preferablyabout 100 percent sequence identity as compared to a reference sequence.Homology can be determined using, for example, a “BLASTN” algorithm. Itis understood that homologous sequences can accommodate insertions,deletions and substitutions in the nucleotide sequence. Thus, linearsequences of nucleotides can be essentially identical even if some ofthe nucleotide residues do not precisely correspond or align. Thereference sequence may be a subset of a larger sequence, such as aportion of a gene or flanking sequence, or a repetitive portion of achromosome.

As used herein, a “variant” of a glucagon receptor is defined as anamino acid sequence that is different by one or more amino acidsubstitutions. The variant may have “conservative” changes, wherein asubstituted amino acid has similar structural or chemical properties,e.g., replacement of a leucine with isoleucine. More rarely, a variantmay have “nonconservative” changes, e.g., replacement of a glycine witha tryptophan. Similar minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which and howmany amino acid residues may be substituted, inserted or deleted withoutabolishing biological or immunological activity may be found usingcomputer programs well known in the art, for example, DNAStar software.

The term “active fragment” refers to a fragment of a glucagon receptorthat is biologically or immunologically active. The term “biologicallyactive” refers to a glucagon receptor having structural, regulatory orbiochemical functions of the naturally occurring glucagon receptor.Likewise, “immunologically active” defines the capability of thenatural, recombinant or synthetic glucagon receptor, or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies.

The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid sequence encoding a glucagon receptor orthe encoded glucagon receptor. An example of such modifications would bereplacement of hydrogen by an alkyl, acyl, or amino group. A nucleicacid derivative would encode a polypeptide which retains essentialbiological characteristics of a natural glucagon receptor.

The term “target gene” (alternatively referred to as “target genesequence” or “target DNA sequence” or “target sequence”) refers to anynucleic acid molecule, polynucleotide, or gene to be modified byhomologous recombination. The target sequence includes an intact gene,an exon or intron, a regulatory sequence or any region between genes.The target gene may comprise a portion of a particular gene or geneticlocus in the individual's genomic DNA. As provided herein, the targetgene of the present invention comprises the glucagon receptor gene asidentified and shown in SEQ ID NO: 1 or as identified and shown inGenbank Accession No.: L38613; GI No.: 603463 or to any derivatives,homologues, mutants or fragments of these sequences. In one aspect, theglucagon receptor gene encodes the glucagon receptor comprising theamino acid sequence identified herein as SEQ ID NO:2 or as identifiedand shown in Genbank Accession No. AAA88244; GI No.: 603464 or anyderivatives, homologues, mutants or fragments of these sequences.

“Disruption” of a glucagon receptor gene occurs when a fragment ofgenomic DNA locates and recombines with an endogenous homologoussequence. These sequence disruptions or modifications may includeinsertions, missense, frameshift, deletion, or substitutions, orreplacements of DNA sequence, or any combination thereof. Insertionsinclude the insertion of entire genes, which may be of animal, plant,fungal, insect, prokaryotic, or viral origin. Disruption, for example,can alter or replace a promoter, enhancer, or splice site of a glucagonreceptor gene, and can alter the normal gene product by inhibiting itsproduction partially or completely or by enhancing the normal geneproduct's activity. In a preferred embodiment, the disruption is a nulldisruption, wherein there is no significant expression of the glucagonreceptor gene.

The term “transgenic cell” refers to a cell containing within its genomea glucagon receptor gene that has been disrupted, modified, altered, orreplaced completely or partially by the method of gene targeting.

The term “transgenic animal” refers to an animal that contains withinits genome a specific gene that has been disrupted or otherwise modifiedor mutated by the method of gene targeting. “Transgenic animal” includesboth the heterozygous animal (i.e., one defective allele and onewild-type allele) and the homozygous animal (i.e., two defectivealleles).

As used herein, the terms “selectable marker” and “positive selectionmarker” refer to a gene encoding a product that enables only the cellsthat carry the gene to survive and/or grow under certain conditions. Forexample, plant and animal cells that express the introduced neomycinresistance (Neo^(r)) gene are resistant to the compound G418. Cells thatdo not carry the Neo^(r) gene marker are killed by G418. Other positiveselection markers are known to or are within the purview of those ofordinary skill in the art.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) or for incorporation of nucleic acidmolecules and/or proteins. Host cells include progeny of a single hostcell, and the progeny may not necessarily be completely identical (inmorphology or in total DNA complement) to the original parent due tonatural, accidental, or deliberate mutation. A host cell includes cellstransfected with the constructs of the present invention.

The term “modulates” as used herein refers to the decrease, inhibition,reduction, increase or enhancement of glucagon receptor gene function,expression, activity, or alternatively a phenotype associated with adisruption in a glucagon receptor gene.

The term “ameliorates” refers to a decrease, reduction or elimination ofa condition, disease, disorder, or phenotype, including an abnormalityor symptom associated with a disruption in a glucagon receptor gene.

The term “abnormality” refers to any disease, disorder, condition, orphenotype in which a disruption of a glucagon receptor gene isimplicated, including pathological conditions and behavioralobservations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the polynucleotide sequence for a murine glucagon receptorgene (SEQ ID NO: 1). FIG. 1B shows the amino acid sequence for themurine glucagon receptor (SEQ ID NO:2).

FIGS. 2A-2B show the location of the disrupted portion of the glucagonreceptor gene, as well as the nucleotide sequences flanking the Neo^(r)insert in the targeting construct.

FIG. 2B shows the sequences identified as SEQ ID NO:3 and SEQ ID NO:4,which were used as the 5′ and 3′ targeting arms (including thehomologous sequences) in the glucagon receptor targeting construct,respectively.

FIG. 3A shows the polynucleotide sequence for a human glucagon receptorgene (SEQ ID NO:5).

FIG. 3B shows the amino acid sequence for the human glucagon receptor(SEQ ID NO:6).

FIG. 4 shows the fasting whole blood glucose levels in homozygous,heterozygous, and wild-type mice.

FIG. 5 shows data relating to the relationship of blood glucose levelsto the severity of pancreatic lesions.

FIG. 6 shows data relating to serum insulin levels in homozygous,heterozygous, and wild-type mice.

FIG. 7 shows data relating to the relationship of serum insulin levelsto the severity of pancreatic lesions.

FIG. 8 shows the serum glucagon levels in homozygous, heterozygous, andwild-type mice.

FIG. 9 shows the response of homozygous mutant mice, heterozygous mutantmice, and wild-type mice to a glucose challenge (Glucose tolerance test,GTT—before a high-fat diet challenge).

FIG. 10 shows the response of homozygous, heterozygous, and wild-typemice to insulin in the insulin suppression test (IST).

FIG. 11 shows the insulin response of homozygous, heterozygous, andwild-type mice to a glucose load (Glucose-stimulated insulin secretiontest, GSIST).

FIG. 12 shows the body weight gain of homozygous mutant, heterozygousmutant, and wild-type mice after eight weeks on a high-fat diet.

FIG. 13 shows the response of homozygous mutant mice, heterozygousmutant mice, and wild-type mice to a glucose challenge (GTT) after eightweeks on a high-fat diet.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the evaluation of the expression androle of genes and gene expression products, primarily those associatedwith a glucagon receptor gene. Among other uses or applications, theinvention permits the definition of disease pathways and theidentification of diagnostically and therapeutically useful targets. Forexample, genes that are mutated or down-regulated under diseaseconditions may be involved in causing or exacerbating the diseasecondition. Treatments directed at up-regulating the activity of suchgenes or treatments that involve alternate pathways, may ameliorate thedisease condition.

Generation of Targeting Construct

The targeting construct of the present invention may be produced usingstandard methods known in the art. (see, e.g., Sambrook, et al., 1989,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; E. N. Glover (eds.),1985, DNA Cloning: A Practical Approach, Volumes I and II; M. J. Gait(ed.), 1984, Oligonucleotide Synthesis; B. D. Hames & S. J. Higgins(eds.), 1985, Nucleic Acid Hybridization; B. D. Hames & S. J. Higgins(eds.), 1984, Transcription and Translation; R. I. Freshney (ed.), 1986,Animal Cell Culture; Immobilized Cells and Enzymes, IRL Press, 1986; B.Perbal, 1984, A Practical Guide To Molecular Cloning; F. M. Ausubel etal., 1994, Current Protocols in Molecular Biology, John Wiley & Sons,Inc.). For example, the targeting construct may be prepared inaccordance with conventional ways, where sequences may be synthesized,isolated from natural sources, manipulated, cloned, ligated, subjectedto in vitro mutagenesis, primer repair, or the like. At various stages,the joined sequences may be cloned, and analyzed by restrictionanalysis, sequencing, or the like.

The targeting DNA can be constructed using techniques well known in theart. For example, the targeting DNA may be produced by chemicalsynthesis of oligonucleotides, nick-translation of a double-stranded DNAtemplate, polymerase chain-reaction amplification of a sequence (orligase chain reaction amplification), purification of prokaryotic ortarget cloning vectors harboring a sequence of interest (e.g., a clonedcDNA or genomic DNA, synthetic DNA or from any of the aforementionedcombination) such as plasmids, phagemids, YACs, cosmids, bacteriophageDNA, other viral DNA or replication intermediates, or purifiedrestriction fragments thereof, as well as other sources of single anddouble-stranded polynucleotides having a desired nucleotide sequence.Moreover, the length of homology may be selected using known methods inthe art. For example, selection may be based on the sequence compositionand complexity of the predetermined endogenous target DNA sequence(s).

The targeting construct of the present invention typically comprises afirst sequence homologous to a portion or region of the glucagonreceptor gene and a second sequence homologous to a second portion orregion of the glucagon receptor gene. The targeting construct mayfurther comprise a positive selection marker, which is preferablypositioned in between the first and the second homologous sequences. Thepositive selection marker may be operatively linked to a promoter and apolyadenylation signal.

Other regulatory sequences known in the art may be incorporated into thetargeting construct to disrupt or control expression of a particulargene in a specific cell type. In addition, the targeting construct mayalso include a sequence coding for a screening marker, for example,green fluorescent protein (GFP), or another modified fluorescentprotein.

Although the size of the homologous sequence is not critical and canrange from as few as about 15-20 base pairs to as many as 100 kb,preferably each fragment is greater than about 1 kb in length, morepreferably between about 1 and about 10 kb, and even more preferablybetween about 1 and about 5 kb. One of skill in the art will recognizethat although larger fragments may increase the number of homologousrecombination events in ES cells, larger fragments will also be moredifficult to clone.

In a preferred embodiment of the present invention, the targetingconstruct is prepared directly from a plasmid genomic library using themethods described in pending U.S. patent application Ser. No.08/971,310, filed Nov. 17, 1997, the disclosure of which is incorporatedherein in its entirety. Generally, a sequence of interest is identifiedand isolated from a plasmid library in a single step using, for example,long-range PCR. Following isolation of this sequence, a secondpolynucleotide that will disrupt the target sequence can be readilyinserted between two regions encoding the sequence of interest. Inaccordance with this aspect, the construct is generated in two steps by(1) amplifying (for example, using long-range PCR) sequences homologousto the target sequence, and (2) inserting another polynucleotide (forexample a selectable marker) into the PCR product so that it is flankedby the homologous sequences. Typically, the vector is a plasmid from aplasmid genomic library. The completed construct is also typically acircular plasmid.

In another embodiment, the targeting construct is designed in accordancewith the regulated positive selection method described in U.S. patentapplication Ser. No. 09/954,483, filed Sep. 17, 2000, the disclosure ofwhich is incorporated herein in its entirety. The targeting construct isdesigned to include a PGK-neo fusion gene having two lacO sites,positioned in the PGK promoter and an NLS-lacl gene comprising a lacrepressor fused to sequences encoding the NLS from the SV40 T antigen.

In another embodiment, the targeting construct may contain more than oneselectable maker gene, including a negative selectable marker, such asthe herpes simplex virus tk (HSV-tk) gene. The negative selectablemarker may be operatively linked to a promoter and a polyadenylationsignal. (see, e.g., U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; and5,631,153).

Generation of Cells and Confirmation of Homologous Recombination Events

Once an appropriate targeting construct has been prepared, the targetingconstruct may be introduced into an appropriate host cell using anymethod known in the art. Various techniques may be employed in thepresent invention, including, for example: pronuclear microinjection;retrovirus mediated gene transfer into germ lines; gene targeting inembryonic stem cells; electroporation of embryos; sperm-mediated genetransfer; and calcium phosphate/DNA co-precipitates, microinjection ofDNA into the nucleus, bacterial protoplast fusion with intact cells,transfection, polycations, e.g., polybrene, polyomithine, etc., or thelike (see, e.g., U.S. Pat. No. 4,873,191; Van der Putten, et al., 1985,Proc. Natl. Acad. Sci., USA 82:6148-6152; Thompson, et al., 1989, Cell56:313-321; Lo, 1983, Mol Cell. Biol. 3:1803-1814; Lavitrano, etal.,1989, Cell, 57:717-723). Various techniques for transformingmammalian cells are known in the art. (see, e.g., Gordon, 1989, Intl.Rev. Cytol., 115:171-229; Keown et al., 1989, Methods in Enzymology;Keown et al., 1990, Methods and Enzymology, Vol. 185, pp. 527-537;Mansour et al., 1988, Nature, 336:348-352).

In a preferred aspect of the present invention, the targeting constructis introduced into host cells by electroporation. In this process,electrical impulses of high field strength reversibly permeabilizebiomembranes allowing the introduction of the construct. The porescreated during electroporation permit the uptake of macromolecules suchas DNA. (see, e.g., Potter, H., et al., 1984, Proc. Nat'l. Acad. Sci.U.S.A. 81:7161-7165).

Any cell type capable of homologous recombination may be used in thepractice of the present invention. Examples of such target cells includecells derived from vertebrates including mammals such as humans, bovinespecies, ovine species, murine species, simian species, and ethereucaryotic organisms such as filamentous fungi, and higher multicellularorganisms such as plants.

Preferred cell types include embryonic stem (ES) cells, which aretypically obtained from pre-implantation embryos cultured in vitro.(see, e.g., Evans, M. J., et al., 1981, Nature 292:154-156; Bradley, M.O., et al., 1984, Nature 309:255-258; Gossler et al., 1986, Proc. Natl.Acad. Sci. USA 83:9065-9069; and Robertson, et al., 1986, Nature322:445-448). The ES cells are cultured and prepared for introduction ofthe targeting construct using methods well known to the skilled artisan.(see, e.g., Robertson, E. J. ed. “Teratocarcinomas and Embryonic StemCells, a Practical Approach”, IRL Press, Washington D.C., 1987; Bradleyet al., 1986, Current Topics in Devel. Biol. 20:357-371; by Hogan etal., in “Manipulating the Mouse Embryo”: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor N.Y., 1986; Thomas etal., 1987, Cell 51:503; Koller et al., 1991, Proc. Natl. Acad. Sci. USA,88:10730; Dorin et al., 1992, Transgenic Res. 1:101; and Veis et al.,1993, Cell 75:229). The ES cells that will be inserted with thetargeting construct are derived from an embryo or blastocyst of the samespecies as the developing embryo into which they are to be introduced.ES cells are typically selected for their ability to integrate into theinner cell mass and contribute to the germ line of an individual whenintroduced into the mammal in an embryo at the blastocyst stage ofdevelopment. Thus, any ES cell line having this capability is suitablefor use in the practice of the present invention.

The present invention may also be used to knock out or otherwise modifyor disrupt genes in other cell types, such as stem cells. By way ofexample, stem cells may be myeloid, lymphoid, or neural progenitor andprecursor cells. These cells comprising a disruption of a gene may beparticularly useful in the study of glucagon receptor gene function inindividual developmental pathways. Stem cells may be derived from anyvertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human,non-human primates and the like.

After the targeting construct has been introduced into cells, the cellsin which successful gene targeting has occurred are identified.Insertion of the targeting construct into the targeted gene is typicallydetected by identifying cells for expression of the marker gene. In apreferred embodiment, the cells transformed with the targeting constructof the present invention are subjected to treatment with an appropriateagent that selects against cells not expressing the selectable marker.Only those cells expressing the selectable marker gene survive and/orgrow under certain conditions. For example, cells that express theintroduced neomycin resistance gene are resistant to the compound G418,while cells that do not express the neo gene marker are killed by G418.If the targeting construct also comprises a screening marker such asGFP, homologous recombination can be identified through screening cellcolonies under a fluorescent light. Cells that have undergone homologousrecombination will have deleted the GFP gene and will not fluoresce.

If a regulated positive selection method is used in identifyinghomologous recombination events, the targeting construct is designed sothat the expression of the selectable marker gene is regulated in amanner such that expression is inhibited following random integrationbut is permitted (derepressed) following homologous recombination. Moreparticularly, the transfected cells are screened for expression of theneo gene, which requires that (1) the cell was successfullyelectroporated, and (2) lac repressor inhibition of neo transcriptionwas relieved by homologous recombination. This method allows for theidentification of transfected cells and homologous recombinants to occurin one step with the addition of a single drug.

Alternatively, a positive-negative selection technique may be used toselect homologous recombinants. This technique involves a process inwhich a first drug is added to the cell population, for example, aneomycin-like drug to select for growth of transfected cells, i.e.positive selection. A second drug, such as FIAU is subsequently added tokill cells that express the negative selection marker, i.e. negativeselection. Cells that contain and express the negative selection markerare killed by a selecting agent, whereas cells that do not contain andexpress the negative selection marker survive. For example, cells withnon-homologous insertion of the construct express HSV thymidine kinaseand therefore are sensitive to the herpes drugs such as gancyclovir(GANC) or FIAU (1-(2-deoxy2-fluoro-B-D-arabinofluranosyl)-5-iodouracil). (see, e.g., Mansour etal., Nature 336:348-352: (1988); Capecchi, Science 244:1288-1292,(1989); Capecchi, Trends in Genet. 5:70-76 (1989)).

Successful recombination may be identified by analyzing the DNA of theselected cells to confirm homologous recombination. Various techniquesknown in the art, such as PCR and/or Southern analysis may be used toconfirm homologous recombination events.

Homologous recombination may also be used to disrupt genes in stemcells, and other cell types, which are not totipotent embryonic stemcells. By way of example, stem cells may be myeloid, lymphoid, or neuralprogenitor and precursor cells. Such transgenic cells may beparticularly useful in the study of glucagon receptor gene function inindividual developmental pathways. Stem cells may be derived from anyvertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human,non-human primates and the like.

In cells that are not totipotent, it may be desirable to knock out bothcopies of the target using methods that are known in the art. Forexample, cells comprising homologous recombination at a target locusthat have been selected for expression of a positive selection marker(e.g., Neor) and screened for non-random integration, can be furtherselected for multiple copies of the selectable marker gene by exposureto elevated levels of the selective agent (e.g., G418). The cells arethen analyzed for homozygosity at the target locus. Alternatively, asecond construct can be generated with a different positive selectionmarker inserted between the two homologous sequences. The two constructscan be introduced into the cell either sequentially or simultaneously,followed by appropriate selection for each of the positive marker genes.The final cell is screened for homologous recombination of both allelesof the target.

Production of Transgenic Animals

Selected cells are then injected into a blastocyst (or other stage ofdevelopment suitable for the purposes of creating a viable animal, suchas, for example, a morula) of an animal (e.g., a mouse) to form chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed., IRL, Oxford, pp. 113-152(1987)). Alternatively, selected ES cells can be allowed to aggregatewith dissociated mouse embryo cells to form the aggregation chimera. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Chimeric progenyharboring the homologously recombined DNA in their germ cells can beused to breed animals in which all cells of the animal contain thehomologously recombined DNA. In one embodiment, chimeric progeny miceare used to generate a mouse with a heterozygous disruption in theglucagon receptor gene. Heterozygous transgenic mice can then be mated.It is well known in the art that typically ¼ of the offspring of suchmatings will have a homozygous disruption in the glucagon receptor gene.

The heterozygous and homozygous transgenic mice can then be compared tonormal, wild-type mice to determine whether disruption of the glucagonreceptor gene causes phenotypic changes, especially pathologicalchanges. For example, heterozygous and homozygous mice may be evaluatedfor phenotypic changes by physical examination, necropsy, histology,clinical chemistry, complete blood count, body weight, organ weights,and cytological evaluation of bone marrow. Phenotypic changes may alsocomprise behavioral modifications or abnormalities.

In one embodiment, the phenotype (or phenotypic change) associated witha disruption in the glucagon receptor gene is placed into or stored in adatabase. Preferably, the database includes: (i) genotypic data (e.g.,identification of the disrupted gene) and (ii) phenotypic data (e.g.,phenotype(s) resulting from the gene disruption) associated with thegenotypic data. The database is preferably electronic. In addition, thedatabase is preferably combined with a search tool so that the databaseis searchable.

Conditional Transgenic Animals

The present invention further contemplates conditional transgenic orknockout animals, such as those produced using recombination methods.Bacteriophage P1 Cre recombinase and flp recombinase from yeast plasmidsare two non-limiting examples of site-specific DNA recombinase enzymesthat cleave DNA at specific target sites (lox P sites for crerecombinase and frt sites for flp recombinase) and catalyze a ligationof this DNA to a second cleaved site. A large number of suitablealternative site-specific recombinases have been described, and theirgenes can be used in accordance with the method of the presentinvention. Such recombinases include the Int recombinase ofbacteriophage λ (with or without Xis) (Weisberg, R. et al., in LambdaII, (Hendrix, R., et al., Eds.), Cold Spring Harbor Press, Cold SpringHarbor, N.Y., pp. 211-50 (1983), herein incorporated by reference); TpnIand the β-lactamase transposons (Mercier, et al., J. Bacteriol.,172:3745-57 (1990)); the Tn3 resolvase (Flanagan & Fennewald J. Molec.Biol., 206:295-304 (1989); Stark, et al., Cell, 58:779-90 (1989)); theyeast recombinases (Matsuzaki, et al., J. Bacteriol., 172:610-18(1990)); the B. subtilis SpoIVC recombinase (Sato, et al., J. Bacteriol.172:1092-98 (1990)); the Flp recombinase (Schwartz & Sadowski, J.Molec.Biol., 205:647-658 (1989); Parsons, et al., J. Biol. Chem.,265:4527-33 (1990); Golic & Lindquist, Cell, 59:499-509 (1989); Amin, etal., J. Molec. Biol., 214:55-72 (1990)); the Hin recombinase (Glasgow,et al., J. Biol. Chem., 264:10072-82 (1989)); immunoglobulinrecombinases (Malynn, et al., Cell, 54:453-460 (1988)); and the Cinrecombinase (Haffter & Bickle, EMBO J., 7:3991-3996 (1988); Hubner, etal., J. Molec. Biol., 205:493-500 (1989)), all herein incorporated byreference. Such systems are discussed by Echols (J. Biol. Chem.265:14697-14700 (1990)); de Villartay (Nature, 335:170-74 (1988));Craig, (Ann. Rev. Genet., 22:77-105 (1988)); Poyart-Salmeron, et al.,(EMBO J. 8:2425-33 (1989)); Hunger-Bertling, et al.,(Mol Cell. Biochem.,92:107-16 (1990)); and Cregg & Madden (Mol. Gen. Genet., 219:320-23(1989)), all herein incorporated by reference.

Cre has been purified to homogeneity, and its reaction with the loxPsite has been extensively characterized (Abremski & Hess J. Mol. Biol.259:1509-14 (1984), herein incorporated by reference). Cre protein has amolecular weight of 35,000 and can be obtained commercially from NewEngland Nuclear/Du Pont. The cre gene (which encodes the Cre protein)has been cloned and expressed (Abremski, et al., Cell 32:1301-11 (1983),herein incorporated by reference). The Cre protein mediatesrecombination between two loxP sequences (Sternberg, et al., Cold SpringHarbor Symp. Quant. Biol. 45:297-309 (1981)), which may be present onthe same or different DNA molecule. Because the internal spacer sequenceof the loxP site is asymmetrical, two loxP sites can exhibitdirectionality relative to one another (Hoess & Abremski Proc. Natl.Acad. Sci. U.S.A. 81:1026-29 (1984)). Thus, when two sites on the sameDNA molecule are in a directly repeated orientation, Cre will excise theDNA between the sites (Abremski, et al., Cell 32:1301-11 (1983)).However, if the sites are inverted with respect to each other, the DNAbetween them is not excised after recombination but is simply inverted.Thus, a circular DNA molecule having two loxP sites in directorientation will recombine to produce two smaller circles, whereascircular molecules having two loxP sites in an inverted orientationsimply invert the DNA sequences flanked by the loxP sites. In addition,recombinase action can result in reciprocal exchange of regions distalto the target site when targets are present on separate DNA molecules.

Recombinases have important application for characterizing gene functionin knockout models. When the constructs described herein are used todisrupt glucagon receptor genes, a fusion transcript can be producedwhen insertion of the positive selection marker occurs downstream (3′)of the translation initiation site of the glucagon receptor gene. Thefusion transcript could result in some level of protein expression withunknown consequence. It has been suggested that insertion of a positiveselection marker gene can affect the expression of nearby genes. Theseeffects may make it difficult to determine gene function after aknockout event since one could not discern whether a given phenotype isassociated with the inactivation of a gene, or the transcription ofnearby genes. Both potential problems are solved by exploitingrecombinase activity. When the positive selection marker is flanked byrecombinase sites in the same orientation, the addition of thecorresponding recombinase will result in the removal of the positiveselection marker. In this way, effects caused by the positive selectionmarker or expression of fusion transcripts are avoided.

In one embodiment, purified recombinase enzyme is provided to the cellby direct microinjection. In another embodiment, recombinase isexpressed from a co-transfected construct or vector in which therecombinase gene is operably linked to a functional promoter. Anadditional aspect of this embodiment is the use of tissue-specific orinducible recombinase constructs that allow the choice of when and whererecombination occurs. One method for practicing the inducible forms ofrecombinase-mediated recombination involves the use of vectors that useinducible or tissue-specific promoters or other gene regulatory elementsto express the desired recombinase activity. The inducible expressionelements are preferably operatively positioned to allow the induciblecontrol or activation of expression of the desired recombinase activity.Examples of such inducible promoters or other gene regulatory elementsinclude, but are not limited to, tetracycline, metallothionine,ecdysone, and other steroid-responsive promoters, rapamycin responsivepromoters, and the like (No, et al., Proc. Natl. Acad. Sci. USA,93:3346-51 (1996); Furth, et al., Proc. Natl. Acad. Sci. USA, 91:9302-6(1994)). Additional control elements that can be used include promotersrequiring specific transcription factors such as viral, promoters.Vectors incorporating such promoters would only express recombinaseactivity in cells that express the necessary transcription factors.

Models for Disease

The cell- and animal-based systems described herein can be utilized asmodels for diseases. Animals of any species, including, but not limitedto, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate disease animal models. In addition, cells from humans may beused. These systems may be used in a variety of applications. Suchassays may be utilized as part of screening strategies designed toidentify agents, such as compounds that are capable of amelioratingdisease symptoms. Thus, the animal- and cell-based models may be used toidentify drugs, pharmaceuticals, therapies and interventions that may beeffective in treating disease.

Cell-based systems may be used to identify compounds that may act toameliorate disease symptoms. For example, such cell systems may beexposed to a compound suspected of exhibiting an ability to amelioratedisease symptoms, at a sufficient concentration and for a timesufficient to elicit such an amelioration of disease symptoms in theexposed cells. After exposure, the cells are examined to determinewhether one or more of the disease cellular phenotypes has been alteredto resemble a more normal or more wild-type, non-disease phenotype.

In addition, animal-based disease systems, such as those describedherein, may be used to identify compounds capable of amelioratingdisease symptoms. Such animal models may be used as test substrates forthe identification of drugs, pharmaceuticals, therapies, andinterventions that may be effective in treating a disease or otherphenotypic characteristic of the animal. For example, animal models maybe exposed to a compound or agent suspected of exhibiting an ability toameliorate disease symptoms, at a sufficient concentration and for atime sufficient to elicit such an amelioration of disease symptoms inthe exposed animals. The response of the animals to the exposure may bemonitored by assessing the reversal of disorders associated with thedisease. Exposure may involve treating mother animals during gestationof the model animals described herein, thereby exposing embryos orfetuses to the compound or agent that may prevent or ameliorate thedisease or phenotype. Neonatal, juvenile, and adult animals can also beexposed.

More particularly, using the animal models of the invention, methods ofidentifying agents are provided, in which such agents can be identifiedon the basis of their ability to affect at least one phenotypeassociated with a disruption in a glucagon receptor gene. In oneembodiment, the present invention provides a method of identifyingagents having an effect on glucagon receptor gene expression orfunction. The method includes measuring a physiological response of theanimal, for example, to the agent and comparing the physiologicalresponse of such animal to a control animal, wherein the physiologicalresponse of the animal comprising a disruption in a glucagon receptorgene as compared to the control animal indicates the specificity of theagent. A “physiological response” is any biological or physicalparameter of an animal that can be measured. Molecular assays (e.g.,gene transcription, protein production and degradation rates), physicalparameters (e.g., exercise physiology tests, measurement of variousparameters of respiration, measurement of heart rate or blood pressure,and measurement of bleeding time), behavioral testing, and cellularassays (e.g.,. immunohistochemical assays of cell surface markers, orthe ability of cells to aggregate or proliferate) can be used to assessa physiological response.

The transgenic animals and cells of the present invention may beutilized as models for diseases, disorders, or conditions associatedwith phenotypes relating to a disruption in a glucagon receptor gene. Inone aspect, the phenotype associated with a homozygous or heterozygousdisruption in a gene encoding a glucagon receptor is an abnormality inIslet of Langerhans cells of the pancreas, e.g. hyperplasia,hypertrophy, increased cytoplasmic vacuolization and granularity and/oradenomas of the islet cells, as described in Example 3 set forth below.In another aspect, the phenotype associated with a disruption in a geneencoding a glucagon receptor is one or more of the followingabnormalities: reduced body weight and/or organ weight, abnormal bodyshape (e.g. dwarfism), decreased body fat percentage, decreased serumglucose levels, and infertility (see Example 3 below). In anotheraspect, the transgenic animals and cells of the present inventionexhibit an increased tolerance to glucose as compared to wild-type mice.As set forth in the Example 4 below, the transgenic animals or cells ofthe present invention may serve as models for glucose metabolism.Alternatively, overexpression of the glucagon receptor in a transgenicmodel may serve as a model for diabetes, in particular, type IIdiabetes.

The present invention provides a unique animal model for testing anddeveloping new treatments relating to the behavioral phenotypes.Analysis of the behavioral phenotype allows for the development of ananimal model useful for testing, for instance, the efficacy of proposedgenetic and pharmacological therapies for human genetic diseases, suchas neurological, neuropsychological, or psychotic illnesses.

A statistical analysis of the various behaviors measured can be carriedout using any conventional statistical program routinely used by thoseskilled in the art (such as, for example, “Analysis of Variance” orANOVA). A “p” value of about 0.05 or less is generally considered to bestatistically significant, although slightly higher p values may stillbe indicative of statistically significant differences. To statisticallyanalyze abnormal behavior, a comparison is made between the behavior ofa transgenic animal (or a group thereof) to the behavior of a wild-typemouse (or a group thereof), typically under certain prescribedconditions. “Abnormal behavior” as used herein refers to behaviorexhibited by an animal having a disruption in the glucagon receptorgene, e.g. transgenic animal, which differs from an animal without adisruption in the glucagon receptor gene, e.g. wild-type mouse. Abnormalbehavior consists of any number of standard behaviors that can beobjectively measured (or observed) and compared. In the case ofcomparison, it is preferred that the change be statistically significantto confirm that there is indeed a meaningful behavioral differencebetween the knockout animal and the wild-type control animal. Examplesof behaviors that may be measured or observed include, but are notlimited to, ataxia, rapid limb movement, eye movement, breathing, motoractivity, cognition, emotional behaviors, social behaviors,hyperactivity, hypersensitivity, anxiety, impaired learning, abnormalreward behavior, and abnormal social interaction, such as aggression.

A series of tests may be used to measure the behavioral phenotype of theanimal models of the present invention, including neurological andneuropsychological tests to identify abnormal behavior. These tests maybe used to measure abnormal behavior relating to, for example, learningand memory, eating, pain, aggression, sexual reproduction, anxiety,depression, schizophrenia, and drug abuse. (see, e.g., Crawley & Paylor,Hormones and Behavior 31:197-211 (1997)).

The social interaction test involves exposing a mouse to other animalsin a variety of settings. The social behaviors of the animals (e.g.,touching, climbing, sniffing, and mating) are subsequently evaluated.Differences in behaviors can then be statistically analyzed and compared(see, e.g., S. E. File, et al., Pharmacol. Bioch. Behav. 22:941-944(1985); R. R. Holson, Phys. Behav. 37:239-247 (1986)). Examplarybehavioral tests include the following.

The mouse startle response test typically involves exposing the animalto a sensory (typically auditory) stimulus and measuring the startleresponse of the animal (see, e.g., M. A. Geyer, et al., Brain Res. Bull.25:485-498 (1990); Paylor and Crawley, Psychopharmacology 132:169-180(1997)). A pre-pulse inhibition test can also be used, in which thepercent inhibition (from a normal startle response) is measured by“cueing” the animal first with a brief low-intensity pre-pulse prior tothe startle pulse.

The electric shock test generally involves exposure to an electrifiedsurface and measurement of subsequent behaviors such as, for example,motor activity, learning, social behaviors. The behaviors are measuredand statistically analyzed using standard statistical tests. (see, e.g.,G. J. Kant, et al., Pharm. Bioch. Behav. 20:793-797 (1984); N. J.Leidenheimer, et al., Pharmacol. Bioch. Behav. 30:351-355 (1988)).

The tail-pinch or immobilization test involves applying pressure to thetail of the animal and/or restraining the animal's movements. Motoractivity, social behavior, and cognitive behavior are examples of theareas that are measured. (see, e.g., M. Bertolucci D'Angic, et al.,Neurochem. 55:1208-1214 (1990)).

The novelty test generally comprises exposure to a novel environmentand/or novel objects. The animal's motor behavior in the novelenvironment and/or around the novel object are measured andstatistically analyzed. (see, e.g., D. K. Reinstein, et al., Pharm.Bioch. Behav. 17:193-202 (1982); B. Poucet, Behav. Neurosci.103:1009-10016 (1989); R. R. Holson, et al., Phys. Behav. 37:231-238(1986)). This test may be used to detect visual processing deficienciesor defects.

The learned helplessness test involves exposure to stresses, forexample, noxious stimuli, which cannot be affected by the animal'sbehavior. The animal's behavior can be statistically analyzed usingvarious standard statistical tests. (see, e.g., A. Leshner, et al.,Behav. Neural Biol. 26:497-501 (1979)).

Alternatively, a tail suspension test may be used, in which the“immobile” time of the mouse is measured when suspended “upside-down” byits tail. This is a measure of whether the animal struggles, anindicator of depression. In humans, depression is believed to resultfrom feelings of a lack of control over one's life or situation. It isbelieved that a depressive state can be elicited in animals byrepeatedly subjecting them to aversive situations over which they haveno control. A condition of “learned helplessness” is eventually reached,in which the animal will stop trying to change its circumstances andsimply accept its fate. Animals that stop struggling sooner are believedto be more prone to depression. Studies have shown that theadministration of certain antidepressant drugs prior to testingincreases the amount of time that animals struggle before giving up.

The Morris water-maze test comprises learning spatial orientations inwater and subsequently measuring the animal's behaviors, such as, forexample, by counting the number of incorrect choices. The behaviorsmeasured are statistically analyzed using standard statistical tests.(see, e.g., E. M. Spruijt, et al., Brain Res. 527:192-197 (1990)).

Alternatively, a Y-shaped maze may be used (see, e.g., McFarland, D. J.,Pharmacology, Biochemistry and Behavior 32:723-726 (1989); Dellu, F., etal., Neurobiology of Learning and Memory 73:31-48 (2000)). The Y-maze isgenerally believed to be a test of cognitive ability. The dimensions ofeach arm of the Y-maze can be, for example, approximately 40 cm×8 cm×20cm, although other dimensions may be used. Each arm can also have, forexample, sixteen equally spaced photobeams to automatically detectmovement within the arms. At least two different tests can be performedusing such a Y-maze. In a continuous Y-maze paradigm, mice are allowedto explore all three arms of a Y-maze for, e.g., approximately 10minutes. The animals are continuously tracked using photobeam detectiongrids, and the data can be used to measure spontaneous alteration andpositive bias behavior. Spontaneous alteration refers to the naturaltendency of a “normal” animal to visit the least familiar arm of a maze.An alternation is scored when the animal makes two consecutive turns inthe same direction, thus representing a sequence of visits to the leastrecently entered arm of the maze. Position bias determinesegocentrically defined responses by measuring the animal's tendency tofavor turning in one direction over another. Therefore, the test candetect differences in an animal's ability to navigate on the basis ofallocentric or egocentric mechanisms. The two-trial Y-maze memory testmeasures response to novelty and spatial memory based on a free-choiceexploration paradigm. During the first trial (acquisition), the animalsare allowed to freely visit two arms of the Y-maze for, e.g.,approximately 15 minutes. The third arm is blocked off during thistrial. The second trial (retrieval) is performed after an intertrialinterval of, e.g., approximately 2 hours. During the retrieval trial,the blocked arm is opened and the animal is allowed access to all threearms for, e.g., approximately 5 minutes. Data are collected during theretrieval trial and analyzed for the number and duration of visits toeach arm. Because the three arms of the maze are virtually identical,discrimination between novelty and familiarity is dependent on“environmental” spatial cues around the room relative to the position ofeach arm. Changes in arm entry and duration of time spent in the novelarm in a transgenic animal model may be indicative of a role of thatgene in mediating novelty and recognition processes.

The passive avoidance or shuttle box test generally involves exposure totwo or more environments, one of which is noxious, providing a choice tobe learned by the animal. Behavioral measures include, for example,response latency, number of correct responses, and consistency ofresponse. (see, e.g., R. Ader, et al., Psychon. Sci. 26:125-128 (1972);R. R. Holson, Phys. Behav. 37:221-230 (1986)). Alternatively, azero-maze can be used. In a zero-maze, the animals can, for example, beplaced in a closed quadrant of an elevated annular platform having,e.g., 2 open and 2 closed quadrants, and are allowed to explore forapproximately 5 minutes. This paradigm exploits an approach-avoidanceconflict between normal exploratory activity and an aversion to openspaces in rodents. This test measures anxiety levels and can be used toevaluate the effectiveness of anti-anxiolytic drugs. The time spent inopen quadrants versus closed quadrants may be recorded automatically,with, for example, the placement of photobeams at each transition site.

The food avoidance test involves exposure to novel food and objectivelymeasuring, for example, food intake and intake latency. The behaviorsmeasured are statistically analyzed using standard statistical tests.(see, e.g., B. A. Campbell, et al., J. Comp. Physiol. Psychol. 67:15-22(1969)).

The elevated plus-maze test comprises exposure to a maze, without sides,on a platform, the animal's behavior is objectively measured by countingthe number of maze entries and maze learning. The behavior isstatistically analyzed using standard statistical tests. (see, e.g., H.A. Baldwin, et al., Brain Res. Bull, 20:603-606 (1988)).

The stimulant-induced hyperactivity test involves injection of stimulantdrugs (e.g., amphetamines, cocaine, PCP, and the like), and objectivelymeasuring, for example, motor activity, social interactions, cognitivebehavior. The animal's behaviors are statistically analyzed usingstandard statistical tests. (see, e.g., P. B. S. Clarke, et al.,Psychopharmacology 96:511-520 (1988); P. Kuczenski, et al., J.Neuroscience 11:2703-2712 (1991)).

The self-stimulation test generally comprises providing the mouse withthe opportunity to regulate electrical and/or chemical stimuli to itsown brain. Behavior is measured by frequency and pattern ofself-stimulation. Such behaviors are statistically analyzed usingstandard statistical tests. (see, e.g., S. Nassif, et al., Brain Res.,332:247-257 (1985); W. L. Isaac, et al., Behav. Neurosci. 103:345-355(1989)).

The reward test involves shaping a variety of behaviors, e.g., motor,cognitive, and social, measuring, for example, rapidity and reliabilityof behavioral change, and statistically analyzing the behaviorsmeasured. (see, e.g., L. E. Jarrard, et al., Exp. Brain Res. 61:519-530(1986)).

The DRL (differential reinforcement to low rates of responding)performance test involves exposure to intermittent reward paradigms andmeasuring the number of proper responses, e.g., lever pressing. Suchbehavior is statistically analyzed using standard statistical tests.(see, e.g., J. D. Sinden, et al., Behav. Neurosci. 100:320-329 (1986);V. Nalwa, et al., Behav Brain Res. 17:73-76 (1985); and A. J. Nonneman,et al., J. Comp. Physiol. Psych. 95:588-602 (1981)).

The spatial learning test involves exposure to a complex novelenvironment, measuring the rapidity and extent of spatial learning, andstatistically analyzing the behaviors measured. (see, e.g., N. Pitsikas,et al., Pharm. Bioch. Behav. 38:931-934 (1991); B. poucet, et al., BrainRes. 37:269-280 (1990); D. Christie, et al., Brain Res. 37:263-268(1990); and F. Van Haaren, et al., Behav. Neurosci. 102:481-488 (1988)).Alternatively, an open-field (of) test may be used, in which the greaterdistance traveled for a given amount of time is a measure of theactivity level and anxiety of the animal. When the open field is a novelenvironment, it is believed that an approach-avoidance situation iscreated, in which the animal is “torn” between the drive to explore andthe drive to protect itself. Because the chamber is lighted and has noplaces to hide other than the corners, it is expected that a “normal”mouse will spend more time in the corners and around the periphery thanit will in the center where there is no place to hide. “Normal” micewill, however, venture into the central regions as they explore more andmore of the chamber. It can then be extrapolated that especially anxiousmice will spend most of their time in the corners, with relativelylittle or no exploration of the central region, whereas bold (i.e., lessanxious) mice will travel a greater distance, showing little preferencefor the periphery versus the central region.

The visual, somatosensory and auditory neglect tests generally compriseexposure to a sensory stimulus, objectively measuring, for example,orientating responses, and statistically analyzing the behaviorsmeasured. (see, e.g., J. M. Vargo, et al., Exp. Neurol. 102:199-209(1988)).

The consummatory behavior test generally comprises feeding and drinking,and objectively measuring quantity of consumption. The behavior measuredis statistically analyzed using standard statistical tests. (see, e.g.,P. J. Fletcher, et al., Psychopharmacol. 102:301-308 (1990); M. G.Corda, et al.,, Proc. Nat'l Acad. Sci. USA 80:2072-2076 (1983)).

A visual discrimination test can also be used to evaluate the visualprocessing of an animal. One or two similar objects are placed in anopen field and the animal is allowed to explore for about 5-10 minutes.The time spent exploring each object (proximity to, i.e., movementwithin, e.g., about 3-5 cm of the object is considered exploration of anobject) is recorded. The animal is then removed from the open field, andthe objects are replaced by a similar object and a novel object. Theanimal is returned to the open field and the percent time spentexploring the novel object over the old object is measured (again, overabout a 5-10 minute span). “Normal” animals will typically spend ahigher percentage of time exploring the novel object rather than the oldobject. If a delay is imposed between sampling and testing, the memorytask becomes more hippocampal-dependent. If no delay is imposed, thetask is more based on simple visual discrimination. This test can alsobe used for olfactory discrimination, in which the objects (preferably,simple blocks) can be sprayed or otherwise treated to hold an odor. Thistest can also be used to determine if the animal can make gustatorydiscriminations; animals that return to the previously eaten foodinstead of novel food exhibit gustatory neophobia.

A hot plate analgesia test can be used to evaluate an animal'ssensitivity to heat or painful stimuli. For example, a mouse can beplaced on an approximately 55° C. hot plate and the mouse's responselatency (e.g., time to pick up and lick a hind paw) can be recorded.These responses are not reflexes, but rather “higher” responsesrequiring cortical involvement. This test may be used to evaluate anociceptive disorder.

An accelerating rotarod test may be used to measure coordination andbalance in mice. Animals can be, for example, placed on a rod that actslike a rotating treadmill (or rolling log). The rotarod can be made torotate slowly at first and then progressively faster until it reaches aspeed of, e.g., approximately 60 rpm. The mice must continuallyreposition themselves in order to avoid falling off. The animals arepreferably tested in at least three trials, a minimum of 20 minutesapart. Those mice that are able to stay on the rod the longest arebelieved to have better coordination and balance.

A metrazol administration test can be used to screen animals for varyingsusceptibilities to seizures or similar events. For example, a 5mg/mlsolution of metrazol can be infused through the tail vein of a mouse ata rate of, e.g., approximately 0.375 ml/min. The infusion will cause allmice to experience seizures, followed by death. Those mice that enterthe seizure stage the soonest are believed to be more prone to seizures.Four distinct physiological stages can be recorded: soon after the startof infusion, the mice will exhibit a noticeable “twitch”, followed by aseries of seizures, ending in a final tensing of the body known as“tonic extension”, which is followed by death.

Glucagon Receptor Gene Products

The present invention further contemplates use of the glucagon receptorgene sequence to produce glucagon receptor gene products. Glucagonreceptor gene products may include proteins that represent functionallyequivalent gene products. Such an equivalent gene product may containdeletions, additions or substitutions of amino acid residues within theamino acid sequence encoded by the gene sequences described herein, butwhich result in a silent change, thus producing a functionallyequivalent glucagon receptor gene product. Amino acid substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved.

For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Functionally equivalent”, as utilized herein, refers toa protein capable of exhibiting a substantially similar in vivo activityas the endogenous gene products encoded by the glucagon receptor genesequences. Alternatively, when utilized as part of an assay,“functionally equivalent” may refer to peptides capable of interactingwith other cellular or extracellular molecules in a manner substantiallysimilar to the way in which the corresponding portion of the endogenousgene product would.

Other protein products useful according to the methods of the inventionare peptides derived from or based on the glucagon receptor geneproduced by recombinant or synthetic means (derived peptides).

Glucagon receptor gene products may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing the gene polypeptides and peptides of the invention byexpressing nucleic acid encoding gene sequences are described herein.Methods that are well known to those skilled in the art can be used toconstruct expression vectors containing gene protein coding sequencesand appropriate transcriptional/translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination(see, e.g., Sambrook, et al., 1989, supra, and Ausubel, et al., 1989,supra). Alternatively, RNA capable of encoding gene protein sequencesmay be chemically synthesized using, for example, automated synthesizers(see, e.g. Oligonucleotide Synthesis: A Practical Approach, Gait, M. J.ed., IRL Press, Oxford (1984)).

A variety of host-expression vector systems may be utilized to expressthe gene coding sequences of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells that may,when transformed or transfected with the appropriate nucleotide codingsequences, exhibit the gene protein of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing gene proteincoding sequences; yeast (e.g. Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing the gene protein codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the gene proteincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing gene protein coding sequences; ormammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionine promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the geneprotein being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of antibodies or to screenpeptide libraries, for example, vectors that direct the expression ofhigh levels of fusion protein products that are readily purified may bedesirable. Such vectors include, but are not limited, to the E. coliexpression vector pUR278 (Ruther et al., EMBO J., 2:1791-94 (1983)), inwhich the gene protein coding sequence may be ligated individually intothe vector in frame with the lac Z coding region so that a fusionprotein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.,13:3101-09 (1985); Van Heeke et al., J. Biol. Chem., 264:5503-9 (1989));and the like. pGEX vectors may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned glucagon receptor gene protein can be released from theGST moiety.

In a preferred embodiment, full length cDNA sequences are appended within-frame Bam HI sites at the amino terminus and Eco RI sites at thecarboxyl terminus using standard PCR methodologies (Innis, et al. (eds)PCR Protocols: A Guide to Methods and Applications, Academic Press, SanDiego (1990)) and ligated into the pGEX-2TK vector (Pharmacia, Uppsala,Sweden). The resulting cDNA construct contains a kinase recognition siteat the amino terminus for radioactive labeling and glutathioneS-transferase sequences at the carboxyl terminus for affinitypurification (Nilsson, et al., EMBO J., 4: 1075-80 (1985); Zabeau etal., EMBO J., 1: 1217-24 (1982)).

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The gene coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of gene codingsequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed (see, e.g., Smith, et al., J. Virol.46:584-93 (1983); U.S. Pat. No. 4,745,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the gene coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing gene protein in infected hosts. (e.g., see Logan et al.,Proc. Natl. Acad. Sci. USA, 81:3655-59 (1984)). Specific initiationsignals may also be required for efficient translation of inserted genecoding sequences. These signals include the ATG initiation codon andadjacent sequences. In cases where an entire gene, including its owninitiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a portion of thegene coding sequence is inserted, exogenous translational controlsignals, including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBitter, et al., Methods in Enzymol., 153:516-44 (1987)).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellsthat possess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product maybe used. Such mammalian host cells include but are not limited to CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express thegene protein may be engineered. Rather than using expression vectorsthat contain viral origins of replication, host cells can be transformedwith DNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells that stably integrate theplasmid into their chromosomes and grow, to form foci, which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines that express the gene protein. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of the geneprotein.

In a preferred embodiment, timing and/or quantity of expression of therecombinant protein can be controlled using an inducible expressionconstruct. Inducible constructs and systems for inducible expression ofrecombinant proteins will be well known to those skilled in the art.Examples of such inducible promoters or other gene regulatory elementsinclude, but are not limited to, tetracycline, metallothionine,ecdysone, and other steroid-responsive promoters, rapamycin responsivepromoters, and the like (No, et al., Proc. Natl. Acad. Sci. USA,93:3346-51 (1996); Furth, et al., Proc. Natl. Acad. Sci. USA, 91:9302-6(1994)). Additional control elements that can be used include promotersrequiring specific transcription factors such as viral, particularlyHIV, promoters. In one in embodiment, a Tet inducible gene expressionsystem is utilized. (Gossen et al., Proc. Natl. Acad. Sci. USA,89:5547-51 (1992); Gossen, et al., Science, 268:1766-69 (1995)). TetExpression Systems are based on two regulatory elements derived from thetetracycline-resistance operon of the E. coli Tn10 transposon—thetetracycline repressor protein (TetR) and the tetracycline operatorsequence (tetO) to which TetR binds. Using such a system, expression ofthe recombinant protein is placed under the control of the tetO operatorsequence and transfected or transformed into a host cell. In thepresence of TetR, which is co-transfected into the host cell, expressionof the recombinant protein is repressed due to binding of the TetRprotein to the tetO regulatory element. High-level, regulated geneexpression can then be induced in response to varying concentrations oftetracycline (Tc) or Tc derivatives such as doxycycline (Dox), whichcompete with tetO elements for binding to TetR. Constructs and materialsfor tet inducible gene expression are available commercially fromCLONTECH Laboratories, Inc., Palo Alto, Calif.

When used as a component in an assay system, the gene protein may belabeled, either directly or indirectly, to facilitate detection of acomplex formed between the gene protein and a test substance. Any of avariety of suitable labeling systems may be used including but notlimited to radioisotopes such as ¹²⁵I; enzyme labeling systems thatgenerate a detectable calorimetric signal or light when exposed tosubstrate; and fluorescent labels. Where recombinant DNA technology isused to produce the gene protein for such assay systems, it may beadvantageous to engineer fusion proteins that can facilitate labeling,immobilization and/or detection.

Indirect labeling involves the use of a protein, such as a labeledantibody, which specifically binds to the gene product. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments and fragments produced by a Fab expression library.

Production of Antibodies

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more epitopes. Such antibodies mayinclude, but are not limited to polyclonal antibodies, monoclonalantibodies (mAbs), humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, fragments produced by aFab expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. Such antibodies may beused, for example, in the detection of a glucagon receptor gene in abiological sample, or, alternatively, as a method for the inhibition ofabnormal glucagon receptor gene activity. Thus, such antibodies may beutilized as part of disease treatment methods, and/or may be used aspart of diagnostic techniques whereby patients may be tested forabnormal levels of glucagon receptor gene proteins, or for the presenceof abnormal forms of such proteins.

For the production of antibodies, various host animals may be immunizedby injection with the glucagon receptor gene, its expression product ora portion thereof. Such host animals may include but are not limited torabbits, mice, rats, goats and chickens, to name but a few. Variousadjuvants may be used to increase the immunological response, dependingon the host species, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as a glucagon receptor gene product, or an antigenic functionalderivative thereof. For the production of polyclonal antibodies, hostanimals such as those described above, may be immunized by injectionwith gene product supplemented with adjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler and Milstein, Nature, 256:495-7 (1975); and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor, et al.,Immunology Today, 4:72 (1983); Cote, et al., Proc. Natl. Acad. Sci. USA,80:2026-30 (1983)), and the EBV-hybridoma technique (Cole, et al., inMonoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., New York,pp. 77-96 (1985)). Such antibodies may be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., Proc. Natl. Acad. Sci., 81:6851-6855(1984); Takeda, et al., Nature, 314:452-54 (1985)) by splicing the genesfrom a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a murine mAb and a humanimmunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-26 (1988);Huston, et al., Proc. Natl. Acad. Sci. USA, 85:5879-83 (1988); and Ward,et al., Nature, 334:544-46 (1989)) can be adapted to produce gene-singlechain antibodies. Single chain antibodies are typically formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse, etal., Science, 246:1275-81 (1989)) to allow rapid and easy identificationof monoclonal Fab fragments with the desired specificity.

Screening Methods

The present invention may be employed in a process for screening foragents such as agonists, i.e., agents that bind to and activate glucagonreceptor polypeptides, or antagonists, i.e. inhibit the activity orinteraction of glucagon receptor polypeptides with its ligand. Thus,polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures as knownin the art. Any methods routinely used to identify and screen for agentsthat can modulate receptors may be used in accordance with the presentinvention.

The present invention provides methods for identifying and screening foragents that modulate glucagon receptor expression or function. Moreparticularly, cells that contain and express glucagon receptor genesequences may be used to screen for therapeutic agents. Such cells mayinclude non-recombinant monocyte cell lines, such as U937 (ATCC#CRL-1593), THP-1 (ATCC# TIB-202), and P388D1 (ATCC# TIB-63); endothelialcells such as HUVEC's and bovine aortic endothelial cells (BAEC's); aswell as generic mammalian cell lines such as HeLa cells and COS cells,e.g., COS-7 (ATCC# CRL-1651). Further, such cells may includerecombinant, transgenic cell lines. For example, the transgenic mice ofthe invention may be used to generate cell lines, containing one or morecell types involved in a disease, that can be used as cell culturemodels for that disorder. While cells, tissues, and primary culturesderived from the disease transgenic animals of the invention may beutilized, the generation of continuous cell lines is preferred. Forexamples of techniques that may be used to derive a continuous cell linefrom the transgenic animals, see Small, et al., Mol. Cell Biol.,5:642-48 (1985).

Glucagon receptor gene sequences may be introduced into andoverexpressed in the genome of the cell of interest. In order tooverexpress a glucagon receptor gene sequence, the coding portion of theglucagon receptor gene sequence may be ligated to a regulatory sequencethat is capable of driving gene expression in the cell type of interest.Such regulatory regions will be well known to those of skill in the art,and may be utilized in the absence of undue experimentation. Glucagonreceptor gene sequences may also be disrupted or underexpressed. Cellshaving glucagon receptor gene disruptions or underexpressed glucagonreceptor gene sequences may be used, for example, to screen for agentscapable of affecting alternative pathways that compensate for any lossof function attributable to the disruption or underexpression.

In vitro systems may be designed to identify compounds capable ofbinding the glucagon receptor gene products. Such compounds may include,but are not limited to, peptides made of D-and/or L-configuration aminoacids (in, for example, the form of random peptide libraries; (see e.g.,Lam, et al., Nature, 354:82-4 (1991)), phosphopeptides (in, for example,the form of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, et al., Cell, 72:767-78 (1993)),antibodies, and small organic or inorganic molecules. Compoundsidentified may be useful, for example, in modulating the activity ofglucagon receptors, preferably mutant glucagon receptors; elaboratingthe biological function of the glucagon receptor gene protein; orscreening for compounds that disrupt normal glucagon receptor geneinteractions or themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to theglucagon receptor involves preparing a reaction mixture of the glucagonreceptor and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex that can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoring theglucagon receptor or the test substance onto a solid phase and detectingtarget protein/test substance complexes anchored on the solid phase atthe end of the reaction. In one embodiment of such a method, theglucagon receptor may be anchored onto a solid surface, and the testcompound, which is not anchored, may be labeled, either directly orindirectly.

In practice, microtitre plates are conveniently utilized. The anchoredcomponent may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of the protein and drying. Alternatively, animmobilized antibody, preferably a monoclonal antibody, specific for theprotein may be used to anchor the protein to the solid surface. Thesurfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for the glucagonreceptor gene product or the test compound to anchor any complexesformed in solution, and a labeled antibody specific for the othercomponent of the possible complex to detect anchored complexes.

Compounds that are shown to bind to a particular glucagon receptor geneproduct through one of the methods described above can be further testedfor their ability to elicit a biochemical response from the glucagonreceptor gene protein. Agonists, antagonists and/or inhibitors of theexpression product can be identified utilizing assays well known in theart.

Antisense, Ribozymes, and Antibodies

Other agents that may be used as therapeutics include the glucagonreceptor gene, its expression product(s) and functional fragmentsthereof. Additionally, agents that reduce or inhibit mutant glucagonreceptor gene activity may be used to ameliorate disease symptoms. Suchagents include antisense, ribozyme, and triple helix molecules.Techniques for the production and use of such molecules are well knownto those of skill in the art.

Anti-sense RNA and DNA molecules act to directly block the translationof mRNA by hybridizing to targeted mRNA and preventing proteintranslation. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between the −10 and+10 regions of the glucagon receptor gene nucleotide sequence ofinterest, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage. Thecomposition of ribozyme molecules must include one or more sequencescomplementary to the glucagon receptor gene mRNA, and must include thewell known catalytic sequence responsible for mRNA cleavage. For thissequence, see U.S. Pat. No. 5,093,246, which is incorporated byreference herein in its entirety. As such within the scope of theinvention are engineered hammerhead motif ribozyme molecules thatspecifically and efficiently catalyze endonucleolytic cleavage of RNAsequences encoding glucagon receptor gene proteins.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the molecule of interest for ribozymecleavage sites that include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the glucagon receptorgene containing the cleavage site may be evaluated for predictedstructural features, such as secondary structure, that may render theoligonucleotide sequence unsuitable. The suitability of candidatesequences may also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays.

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription should be single stranded and composed ofdeoxyribonucleotides. The base composition of these oligonucleotidesmust be designed to promote triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of eitherpurines or pyrimidines to be present on one strand of a duplex.Nucleotide sequences may be pyrimidine-based, which will result in TATand CGC triplets across the three associated strands of the resultingtriple helix. The pyrimidine-rich molecules provide base complementarityto a purine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

It is possible that the antisense, ribozyme, and/or triple helixmolecules described herein may reduce or inhibit the transcription(triple helix) and/or translation (antisense, ribozyme) of mRNA producedby both normal and mutant glucagon receptor gene alleles. In order toensure that substantially normal levels of glucagon receptor geneactivity are maintained, nucleic acid molecules that encode and expressglucagon receptor polypeptides exhibiting normal activity may beintroduced into cells that do not contain sequences susceptible towhatever antisense, ribozyme, or triple helix treatments are beingutilized. Alternatively, it may be preferable to coadminister normalglucagon receptor protein into the cell or tissue in order to maintainthe requisite level of cellular or tissue glucagon receptor geneactivity.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various well-known modifications to the DNA molecules may be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include but are not limited to the addition of flankingsequences of ribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ends of the molecule or the use of phosphorothioate or 2′ O-methylrather than phosphodiesterase linkages within theoligodeoxyribonucleotide backbone.

Antibodies that are both specific for the glucagon receptor protein, andin particular, the mutant glucagon receptor protein, and interfere withits activity may be used to inhibit mutant glucagon receptor genefunction. Such antibodies may be generated against the proteinsthemselves or against peptides corresponding to portions of the proteinsusing standard techniques known in the art and as also described herein.Such antibodies include but are not limited to polyclonal, monoclonal,Fab fragments, single chain antibodies, chimeric antibodies, antibodymimetics, etc.

In instances where the glucagon receptor protein is intracellular andwhole antibodies are used, internalizing antibodies may be preferred.However, lipofectin liposomes may be used to deliver the antibody or afragment of the Fab region that binds to the glucagon receptor geneepitope into cells. Where fragments of the antibody are used, thesmallest inhibitory fragment that binds to the target or expanded targetprotein's binding domain is preferred. For example, peptides having anamino acid sequence corresponding to the domain of the variable regionof the antibody that binds to the glucagon receptor protein may be used.Such peptides may be synthesized chemically or produced via recombinantDNA technology using methods well known in the art (see, e.g.,Creighton, Proteins: Structures and Molecular Principles (1984) W. H.Freeman, New York 1983, supra; and Sambrook, et al., 1989, supra).Alternatively, single chain neutralizing antibodies that bind tointracellular glucagon receptor gene epitopes may also be administered.Such single chain antibodies may be administered, for example, byexpressing nucleotide sequences encoding single-chain antibodies withinthe target cell population by utilizing, for example, techniques such asthose described in Marasco, et al., Proc. Natl. Acad. Sci. USA,90:7889-93 (1993).

RNA sequences encoding the glucagon receptor protein may be directlyadministered to a patient exhibiting disease symptoms, at aconcentration sufficient to produce a level of glucagon receptor proteinsuch that disease symptoms are ameliorated. Patients may be treated bygene replacement therapy. One or more copies of a normal glucagonreceptor gene, or a portion of the gene that directs the production of anormal glucagon receptor protein with glucagon receptor gene function,may be inserted into cells using vectors that include, but are notlimited to adenovirus, adeno-associated virus, and retrovirus vectors,in addition to other particles that introduce DNA into cells, such asliposomes. Additionally, techniques such as those described above may beutilized for the introduction of normal glucagon receptor gene sequencesinto human cells.

Cells, preferably autologous cells, containing normal glucagon receptorgene expressing gene sequences may then be introduced or reintroducedinto the patient at positions that allow for the amelioration of diseasesymptoms.

Pharmaceutical Compositions, Effective Dosages, and Routes ofAdministration

The identified compounds that inhibit target mutant gene expression,synthesis and/or activity can be administered to a patient attherapeutically effective doses to treat or ameliorate the disease. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of the disease.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by inhalation or insufflation (eitherthrough the mouth or the nose) or oral, buccal, parenteral, topical,subcutaneous, intraperitoneal, intraveneous, intrapleural, intraoccular,intraarterial, or rectal administration. It is also contemplated thatpharmaceutical compositions may be administered with other products thatpotentiate the activity of the compound and optionally, may includeother therapeutic ingredients.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. Oralingestion is possibly the easiest method of taking any medication. Sucha route of administration, is generally simple and straightforward andis frequently the least inconvenient or unpleasant route ofadministration from the patient's point of view. However, this involvespassing the material through the stomach, which is a hostile environmentfor many materials, including proteins and other biologically activecompositions. As the acidic, hydrolytic and proteolytic environment ofthe stomach has evolved efficiently to digest proteinaceous materialsinto amino acids and oligopeptides for subsequent anabolism, it ishardly surprising that very little or any of a wide variety ofbiologically active proteinaceous material, if simply taken orally,would survive its passage through the stomach to be taken up by the bodyin the small intestine. The result, is that many proteinaceousmedicaments must be taken in through another method, such asparenterally, often by subcutaneous, intramuscular or intravenousinjection.

Pharmaceutical compositions may also include various buffers (e.g.,Tris, acetate, phosphate), solubilizers (e.g., Tween, Polysorbate),carriers such as human serum albumin, preservatives (thimerosol, benzylalcohol) and anti-oxidants such as ascorbic acid in order to stabilizepharmaceutical activity. The stabilizing agent may be a detergent, suchas tween-20, tween-80, NP-40 or Triton X-100. EBP may also beincorporated into particulate preparations of polymeric compounds forcontrolled delivery to a patient over an extended period of time. A moreextensive survey of components in pharmaceutical compositions is foundin Remington's Pharmaceutical Sciences, 18th ed., A. R. Gennaro, ed.,Mack Publishing, Easton, Pa. (1990).

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Diagnostics

A variety of methods may be employed to diagnose disease conditionsassociated with the glucagon receptor gene. Specifically, reagents maybe used, for example, for the detection of the presence of glucagonreceptor gene mutations, or the detection of either over- orunder-expression of glucagon receptor gene niRNA.

According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type glucagon receptor gene locus isdetected. In addition, the method can be performed by detecting thewild-type glucagon receptor gene locus and confirming the lack of apredisposition or neoplasia. “Alteration of a wild-type gene”encompasses all forms of mutations including deletions, insertions andpoint mutations in the coding and noncoding regions. Deletions may be ofthe entire gene or only a portion of the gene. Point mutations mayresult in stop codons, frameshift mutations or amino acid substitutions.Somatic mutations are those that occur only in certain tissues, e.g., intumor tissue, and are not inherited in the germline. Germline mutationscan be found in any of a body's tissues and are inherited. If only asingle allele is somatically mutated, an early neoplastic state may beindicated. However, if both alleles are mutated, then a late neoplasticstate may be indicated. The finding of gene mutations thus provides bothdiagnostic and prognostic information. A glucagon receptor gene allelethat is not deleted (e.g., that found on the sister chromosome to achromosome carrying a glucagon receptor gene deletion) can be screenedfor other mutations, such as insertions, small deletions, and pointmutations. Mutations found in tumor tissues may be linked to decreasedexpression of the glucagon receptor gene product. However, mutationsleading to non-functional gene products may also be linked to acancerous state. Point mutational events may occur in regulatoryregions, such as in the promoter of the gene, leading to loss ordiminution of expression of the mRNA. Point mutations may also abolishproper RNA processing, leading to loss of expression of the glucagonreceptor gene product, or a decrease in mRNA stability or translationefficiency.

One test available for detecting mutations in a candidate locus is todirectly compare genomic target sequences from cancer patients withthose from a control population. Alternatively, one could sequencemessenger RNA after amplification, e.g., by PCR, thereby eliminating thenecessity of determining the exon structure of the candidate gene.Mutations from cancer patients falling outside the coding region of theglucagon receptor gene can be detected by examining the non-codingregions, such as introns and regulatory sequences near or within theglucagon receptor gene. An early indication that mutations in noncodingregions are important may come from Northern blot experiments thatreveal messenger RNA molecules of abnormal size or abundance in cancerpatients as compared to control individuals.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specific genenucleic acid or anti-gene antibody reagent described herein, which maybe conveniently used, e.g., in clinical settings, to diagnose patientsexhibiting disease symptoms or at risk for developing disease.

Any cell type or tissue, including brain, cortex, subcortical region,cerebellum, brainstem, olfactory bulb, spinal cord, eye, Harderiangland, heart, lung, liver, pancreas, kidney, spleen, thymus, lymphnodes, bone marrow, skin, gallbladder, urinary bladder, pituitary gland,adrenal gland, salivary gland, skeletal muscle, tongue, stomach, smallintestine, large intestine, cecum, testis, epididymis, seminal vesicle,coagulating gland, prostate gland, ovary, uterus and white fat, in whichthe gene is expressed may be utilized in the diagnostics describedbelow.

DNA or RNA from the cell type or tissue to be analyzed may easily beisolated using procedures that are well known to those in the art.Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, PCR In Situ Hybridization:Protocols and Applications, Raven Press, N.Y. (1992)).

Gene nucleotide sequences, either RNA or DNA, may, for example, be usedin hybridization or amplification assays of biological samples to detectdisease-related gene structures and expression. Such assays may include,but are not limited to, Southern or Northern analyses, restrictionfragment length polymorphism assays, single stranded conformationalpolymorphism analyses, in situ hybridization assays, and polymerasechain reaction analyses. Such analyses may reveal both quantitativeaspects of the expression pattern of the gene, and qualitative aspectsof the gene expression and/or gene composition. That is, such aspectsmay include, for example, point mutations, insertions, deletions,chromosomal rearrangements, and/or activation or inactivation of geneexpression.

Preferred diagnostic methods for the detection of gene-specific nucleicacid molecules may involve for example, contacting and incubatingnucleic acids, derived from the cell type or tissue being analyzed, withone or more labeled nucleic acid reagents under conditions favorable forthe specific annealing of these reagents to their complementarysequences within the nucleic acid molecule of interest. Preferably, thelengths of these nucleic acid reagents are at least 9 to 30 nucleotides.After incubation, all non-annealed nucleic acids are removed from thenucleic acid:fingerprint molecule hybrid. The presence of nucleic acidsfrom the fingerprint tissue that have hybridized, if any such moleculesexist, is then detected. Using such a detection scheme, the nucleic acidfrom the tissue or cell type of interest may be immobilized, forexample, to a solid support such as a membrane, or a plastic surfacesuch as that on a microtitre plate or polystyrene beads. In this case,after incubation, non-annealed, labeled nucleic acid reagents are easilyremoved. Detection of the remaining, annealed, labeled nucleic acidreagents is accomplished using standard techniques well-known to thosein the art.

Alternative diagnostic methods for the detection of gene-specificnucleic acid molecules may involve their amplification, e.g., by PCR(the experimental embodiment set forth in Mullis U.S. Pat. No. 4,683,202(1987)), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA,88:189-93 (1991)), self sustained sequence replication (Guatelli, etal., Proc. Natl. Acad. Sci. USA, 87:1874-78 (1990)), transcriptionalamplification system (Kwoh, et al., Proc. Natl. Acad. Sci. USA,86:1173-77 (1989)), Q-Beta Replicase (Lizardi et al., Bio/Technology,6:1197 (1988)), or any other nucleic acid amplification method, followedby the detection of the amplified molecules using techniques well knownto those of skill in the art. These detection schemes are especiallyuseful for the detection of nucleic acid molecules if such molecules arepresent in very low numbers.

In one embodiment of such a detection scheme, a cDNA molecule isobtained from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). Cell types or tissues fromwhich such RNA may be isolated include any tissue in which wild-typefingerprint gene is known to be expressed, including, but not limited,to brain, cortex, subcortical region, cerebellum, brainstem, olfactorybulb, spinal cord, eye, Harderian gland, heart, lung, liver, pancreas,kidney, spleen, thymus, lymph nodes, bone marrow, skin, gallbladder,urinary bladder, pituitary gland, adrenal gland, salivary gland,skeletal muscle, tongue, stomach, small intestine, large intestine,cecum, testis, epididymis, seminal vesicle, coagulating gland, prostategland, ovary, uterus and white fat. A sequence within the cDNA is thenused as the template for a nucleic acid amplification reaction, such asa PCR amplification reaction, or the like. The nucleic acid reagentsused as synthesis initiation reagents (e.g., primers) in the reversetranscription and nucleic acid amplification steps of this method may bechosen from among the gene nucleic acid reagents described herein. Thepreferred lengths of such nucleic acid reagents are at least 15-30nucleotides. For detection of the amplified product, the nucleic acidamplification may be performed using radioactively or non-radioactivelylabeled nucleotides. Alternatively, enough amplified product may be madesuch that the product may be visualized by standard ethidium bromidestaining or by utilizing any other suitable nucleic acid stainingmethod.

Antibodies directed against wild-type or mutant gene peptides may alsobe used as disease diagnostics and prognostics. Such diagnostic methods,may be used to detect abnormalities in the level of gene proteinexpression, or abnormalities in the structure and/or tissue, cellular,or subcellular location of fingerprint gene protein. Structuraldifferences may include, for example, differences in the size,electronegativity, or antigenicity of the mutant fingerprint geneprotein relative to the normal fingerprint gene protein.

Protein from the tissue or cell type to be analyzed may easily bedetected or isolated using techniques that are well known to those ofskill in the art, including but not limited to western blot analysis.For a detailed explanation of methods for carrying out western blotanalysis, see Sambrook, et al. (1989) supra, at Chapter 18. The proteindetection and isolation methods employed herein may also be such asthose described in Harlow and Lane, for example, (Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988)).

Preferred diagnostic methods for the detection of wild-type or mutantgene peptide molecules may involve, for example, immunoassays whereinfingerprint gene peptides are detected by their interaction with ananti-fingerprint gene-specific peptide antibody.

For example, antibodies, or fragments of antibodies useful in thepresent invention may be used to quantitatively or qualitatively detectthe presence of wild-type or mutant gene peptides. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody (see below) coupled with lightmicroscopic, flow cytometric, or fluorimetric detection. Such techniquesare especially preferred if the fingerprint gene peptides are expressedon the cell surface.

The antibodies (or fragments thereof) useful in the present inventionmay, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of fingerprint genepeptides. In situ detection may be accomplished by removing ahistological specimen from a patient, and applying thereto a labeledantibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the fingerprint gene peptides, butalso their distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays for wild-type, mutant, or expanded fingerprint genepeptides typically comprise incubating a biological sample, such as abiological fluid, a tissue extract, freshly harvested cells, or cellsthat have been incubated in tissue culture, in the presence of adetectably labeled antibody capable of identifying fingerprint genepeptides, and detecting the bound antibody by any of a number oftechniques well known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support that is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled gene-specificantibody. The solid phase support may then be washed with the buffer asecond time to remove unbound antibody. The amount of bound label onsolid support may then be detected by conventional means.

The terms “solid phase support or carrier” are intended to encompass anysupport capable of binding an antigen or an antibody. Well-knownsupports or carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-wild-type or -mutantfingerprint gene peptide antibody may be determined according to wellknown methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

One of the ways in which the gene peptide-specific antibody can bedetectably labeled is by linking the same to an enzyme and using it inan enzyme immunoassay (EIA) (Voller, Ric Clin Lab, 8:289-98 (1978) [“TheEnzyme Linked Immunosorbent Assay (ELISA)”, Diagnostic Horizons 2:1-7,1978, Microbiological Associates Quarterly Publication, Walkersville,Md.]; Voller, et al., J. Clin. Pathol., 31:507-20 (1978); Butler, Meth.Enzymol., 73:482-523 (1981); Maggio (ed.), Enzyme Immunoassay, CRCPress, Boca Raton, Fla. (1980); Ishikawa, et al., (eds.) EnzymeImmunoassay, Igaku-Shoin, Tokyo (1981)). The enzyme that is bound to theantibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietythat can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes that can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild-type,mutant, or expanded peptides through the use of a radioimmunoassay (RIA)(see, e.g., Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986). The radioactive isotope can be detected by such means asthe use of a gamma counter or a scintillation counter or byautoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediamine-tetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Throughout this application, various publications, patents and publishedpatent applications are referred to by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications referenced in this application are hereby incorporated byreference into the present disclosure to more fully describe the stateof the art to which this invention pertains.

The following examples are intended only to illustrate the presentinvention and should in no way be construed as limiting the subjectinvention.

EXAMPLES Example 1 Generation of Mice Comprising Glucagon Receptor GeneDisruptions

To investigate the role of glucagon receptors, disruptions in glucagonreceptor genes were produced by homologous recombination. Specifically,transgenic mice comprising disruptions in glucagon receptor genes werecreated. More particularly, as shown in FIG. 2, a glucagon receptortargeting construct having the ability to disrupt a glucagon receptorgene was created, using as the targeting in the construct theoligonucleotide sequences identified herein as SEQ ID NO:3 or SEQ IDNO:4.

The targeting construct was introduced into ES cells derived from the129/OlaHsd mouse substrain to generate chimeric mice. The F1 mice weregenerated by breeding with C57Bu6 females, and the resultant F1NOheterozygotes were backcrossed to C57BL/6 mice to generate F1N1heterozygotes. The F2N1 homozygous mutant mice were produced byintercrossing F1N1 heterozygous males and females.

The transgenic mice comprising disruptions in glucagon receptor geneswere analyzed for phenotypic changes and expression patterns. Thephenotypes associated with a disruption in the glucagon receptor genewere determined.

Example 2 Expression Analysis

RT-PCR Expression. Total RNA was isolated from the organs or tissuesfrom adult C57BL/6 wild-type mice. RNA was DNaseI treated, and reversetranscribed using random primers. The resulting cDNA was checked for theabsence of genomic contamination using primers specific tonon-transcribed genomic mouse DNA. cDNAs were balanced for concentrationusing HPRT primers. RNA transcripts were detectable in liver, kidney,gall bladder, adrenal gland and salivary gland. RNA transcripts were notdetectable in brain, cortex, subcortical region, cerebellum, brainstem,olfactory bulb, eye, heart, lung, pancreas, spleen, thymus, lymph nodes,bone marrow, skin, urinary bladder, pituitary gland, skeletal muscle,tongue, stomach, small intestine, large intestine, cecum, testis,epididymis, seminal vesicle, coagulating gland, prostate gland, ovaryand uterus.

Example 3 Analysis of Transgenic Mice

Histopathology: When compared to age- and gender-matched wild-typecontrol mice, the transgenic mice comprising disruptions in the glucagonreceptor displayed changes in the Islets of Langerhans cells of thepancreas at about 49 days of age. Specifically, all homozygous mutantfemale mice, three out of four homozygous mutant male mice, and oneheterozygous mutant male mouse displayed mild to moderate hyperplasiaand hypertrophy of the Islet cells, as well as increased cytoplasmicvacuolization and granularity. In addition, it was observed that onehomozygous mutant male mouse displayed thymic atrophy and markedtesticular immaturity, and this mouse was clinically identified as adwarf.

Normal Islets of Langerhans cells are predominantly composed ofinsulin-secreting β-cells, with a narrow rim of glucagon-secretingα-cells, and a scattering of somatostatin-secreting δ-cells.Immunohistochemistry performed on tissues from homozygous animalsdemonstrated that α-cells were increased in number and size, whileβ-cells were reduced in number.

At about 300 days of age, all homozygous mutant mice displayed multiplepancreatic islet cell adenomas. All homozygous mutant mice and onewild-type control (male) mouse displayed islet cell hyperplasia.Increased cytoplasmic vacuolization and granularity was observed in boththe islet cell adenomas and hyperplastic islet cells of the homozygousmutant mice relative to wild-type mice.

Necropsy—Body and Organ Weights: At about 49 days of age, the transgenicmice comprising disruptions in glucagon receptor genes generallydisplayed reduced body weights. In particular, some homozygous mutant(−/−) mice displayed reduced body weights relative to wild-type (+/+)control mice. In addition, some homozygous mutant mice displayed reducedorgan weights. The supporting data is provided in the following Table 1.

TABLE 1 Body and Organ Weights Testes + Kidney Heart Epididy Length BodySpleen Liver Weight Thymus Weight Weight Gender Age at Test (cm) Weight(g) Weight (g) Weight (g) (g) Weight (g) (g) (g) +/+ Female 48 9.5 22.740.115 1.154 0.273 0.084 0.127 Female 48 9.2 21.424 0.083 1.155 0.2680.078 0.109 Male 48 9.5 27.705 0.088 1.451 0.408 0.081 0.147 0.259 Male49 9 27.979 0.096 1.542 0.415 0.079 0.162 0.312 −/+ Female 48 9.5 22.290.086 1.139 0.291 0.11 0.109 Female 47 8.25 16.377 0.073 1.042 0.220.078 0.087 Female 47 8.5 18.128 0.071 0.991 0.215 0.104 0.096 Female 478 18.838 0.072 1.086 0.254 0.09 0.101 Male 49 9.25 28.522 0.085 1.7130.409 0.079 0.154 0.252 Male 49 9 23.867 0.098 1.488 0.332 0.059 0.1410.239 Male 49 9.75 26.348 0.12 1.698 0.357 0.087 0.162 0.216 Male 48 9.527.488 0.101 1.596 0.381 0.094 0.194 0.285 −/− Female 48 7.25 9.7150.019 0.344 0.225 0.01 0.057 Female 48 8.5 20.1 0.076 1.204 0.285 0.0680.112 Female 48 9.5 22.034 0.086 1.406 0.343 0.08 0.124 Male 48 9.422.127 0.066 1.174 0.314 0.061 0.121 0.258 Male 48 8.5 18.598 0.066 0.910.368 0.058 0.116 0.169 Male 54 9.5 24.785 0.086 1.563 0.443 0.073 0.1350.283 Male 48 7.5 8.556 0.03 0.285 0.154 0.012 0.059 0.031 Male 45 5.255.279 0.009 0.177 0.098 0.007 0.048 0.037

Physical Exam: At about 49 days of age, the transgenic mice comprisingdisruptions in glucagon receptor genes exhibited abnormal body shape.Specifically, several homozygous mutant mice were identified as dwarfshaped.

Serum Chemistry: Serum samples from homozygous mutant mice, heterozygousmutant mice, and wild-type control mice were evaluated by a clinicalchemistry panel. At about 49 days of age, transgenic mice comprisingdisruptions in glucagon receptors exhibited reduced serum glucose levelsrelative to wild-type (+/+) mice. Specifically, all homozygous (−/−)mutant female mice, two of three homozygous (−/−) mutant male mice, andone heterozygous (−/+) mutant male mouse displayed reduced serum glucoselevels, as shown in Table 2 below. All animals exhibiting reduced serumglucose levels also displayed significant phenotypic histologicalterations of pancreatic islet cells, as noted above.

TABLE 2 Glucose levels Age at Test Genotype Gender (days) Glucose(mg/dl) +/+ Female 48 253 48 258 +/+ Male 48 263 49 278 −/+ Female 48245 47 202 47 169 −/+ Male 49 190 49 198 49 228 48 214 −/− Female 48 14862 151 49 131 −/− Male 48 278 49 159 49 154

At about 73 days of age, homozygous, heterozygous, and wild-type micewere analyzed for fasting whole blood glucose, serum insulin, and serumglucagon measurements. As shown in FIG. 4, fasting blood glucose wasgreatly decreased in homozygous mutant mice, while heterozygous mutantmice had increased fasting blood glucose levels. The relationshipbetween blood glucose levels and the severity of pancreatic lesions isdemonstrated in FIG. 5. Serum insulin levels were decreased inhomozygous mutant mice, as shown in FIG. 6, and the relationship ofinsulin levels to the severity of pancreatic lesions is shown in FIG. 7.Glucagon levels were greatly elevated in homozygous mutant mice (seeFIG. 8) and these levels also correlated with the severity of thepancreatic lesions.

Densitometry: A PIXImus densitometer, which utilizes Dual Energy X-rayabsorptiometry (DXA), was used to measure fat as a percent of body softtissue in approximately 73 day old mice. Homozygous mutant female andmale mice (−/−) displayed decreased fat as a percent of body soft tissuewhen compared to age- and gender-matched wild-type control mice (+/+),as shown in Table 3 below.

TABLE 3 Fat as a Percent of Body Soft Tissue Gender Age at Test GenotypeMouse (days) fat (% of body weight) +/+ Female 120215 73 14.71 120229 7315.71 +/+ Male 120212 73 12.1 120213 73 13.65 −/+ Male 120210 73 13.03120219 73 13.02 120221 73 14.76 120223 73 17.56 −/− Female 120216 7312.24 120225 73 13.2 120227 73 13.06 120228 73 11.97 −/− Male 120211 7311.32 120220 73 9.99 120222 73 11.64

Fertility: Three homozygous mutant mice of each gender were set up formating one on one at about seven to eight weeks of age. The transgenicmice of the present invention were infertile. Specifically, twohomozygous mutant females set up for mating were not able to have pups,and the remaining female was able to produce only one pup.

Example 4 Role of the Glucagon Receptor in Glucose Tolerance andDiabetes

To reveal the potential contribution of the glucagon receptor gene totype II diabetes and obesity, a series of tests were performed onglucagon deficient mice and wild-type control mice. These proceduresincluded the Glucose Tolerance Test (GTT), the Insulin Suppression Test(IST) and the Glucose-stimulated Insulin Secretion Test (GSIST). Glucosetolerance, as seen in type II diabetes, can be the result of eitherinsulin insensitivity, which is the inability of muscle, fat or livercells to take up glucose in response to insulin, or insulin deficiency,usually the result of pancreatic β-cell dysfunction, or both. Thesetests are meant to measure the ability of the mice to metabolize and/orstore glucose, the sensitivity of blood glucose to exogenous insulin,and insulin secretion in response to glucose.

Materials and Methods: Five homozygous mutant, six heterozygous mutant,and five wild-type male mice, approximately one year old were tested forglucose tolerance, insulin sensitivity, and glucose-stimulated insulinsecretion. Mice were maintained on a 12 hour/12 hour dark/light cycleand were fed mouse chow diet (Harlan Teklad, Madison, Wis.) and water adlibitum. One week prior to the tests, mice were individually housed. Onthe day of testing, mice were fasted for 5 hours prior to measuring thebasal glucose plasma concentration or insulin concentration. Water wasstill provided at will during this fasting period.

Glucose Tolerance Test (GTT): Tail vein blood glucose levels weremeasured before injection by collecting 5 to 10 microliters of bloodfrom the tail tip and using glucometers (Glucometer Elite,BayerCorporation, Mishawaka, Ind.). The glucose values were used fortime t=0. Mice were weighed at t=0 and glucose was then administered byi.p. injection at a dose of 2 grams per kilogram of body weight. Plasmaglucose concentrations were measured at 15, 30, 60, 90, and 120 minutesafter injection by the method used to measure basal (t=0) blood glucose.

Mice were returned to cages with access to food ad libitum for one week,after which the GTT was repeated. Glucose values of both tests wereaveraged for statistical analysis. Pair-wise statistical significancewas established using a Student t-test. Weights and plasma glucoseconcentrations are presented as Mean±SE. Statistical significance isdefined as P<0.05. The glucose levels presented were thought to berepresentative of the ability of the mouse to secrete insulin inresponse to elevated glucose levels and the ability of muscle, liver andadipose tissues to uptake glucose.

Insulin Suppression Test (IST): Tail vein glucose levels and body weightwere measured at t=0 as in the GTT above. Insulin (Humulin R, Eli Lillyand Company, Indianapolis, Ind.) was administered by i.p injection at0.5 Units per kilogram body weight. Plasma glucose levels were measuredat 15, 30, 60, 90, and 120 minutes after insulin injection and presentedas the percent of basal glucose. Glucose levels in this test werethought to be representative of the sensitivity of the mouse to insulin(ability of mouse tissues to uptake glucose in response to insulin).

Glucose-Stimulated Insulin Secretion Test (GSIST): Tail vein bloodsamples were taken before the test to measure serum insulin levels att=0. Glucose was administerd by i.p injection at 2 grams per kilogrammouse body weight. Tail vein blood samples were then collected at 7.5,15, 30, and 60 minutes after the glucose loading. Serum insulin levelswere determined by an ELISA kit (Crystan Chem Inc., Chicago, Ill.).

After all three tests were completed, mice were then submitted to ahigh-fat (42%) diet (Adjusted Calories Diet #88137, Harlan Teklad,Madison, Wis.) for eight weeks. Mouse body weight and food intake aremeasured once weekly. GTT was repeated after the high-fat dietchallenge.

Results: The responses of control (+/+) and glucagon receptor mutant(−/− and −/+) mice to the GTT are shown in FIG. 9. Significantdifferences in plasma glucose concentrations were observed in homozygousmutant mice when compared to heterozygous mutant and wild-type mice atall time points after glucose injection (p≦0.03 for all time-points). Itis also noted that fasting glucose levels are decreased in thehomozygous mutant mice and heterozygous mutant mice when compared towild-type mice. These results are indicative that glucagon receptordeficient mice were more tolerant to a glucose challenge relative towild-type and heterozygous mutant mice, and that homozygous andheterozygous mutant mice have lower fasting glucose levels.

As shown in FIG. 10, no difference in glucose levels was seen betweenhomozygous mutant (−/−), heterozygous mutant (−/+), and wild-type mice(+/+) after injection with insulin, indicating that there is not asignificant difference in the sensitivity of blood glucose in theseglucagon receptor deficient mice to exogenous insulin. This suggeststhat it is possible that increased insulin sensitivity is not themechanism for the increased glucose tolerance in the homozygous mutantmice. The results of the GSIST, shown in FIG. 11, show that thehomozygous mutant mice (−/−) have lower fasting insulin levels whencompared to wild-type mice (+/+), with no difference detected at 15minutes post glucose injection, indicating the possibility thatincreased insulin levels are also not the mechanism for the increasedglucose tolerance in glucagon receptor deficient mice.

After exposure to a high fat diet, homozygous mutants (−/−) gain lessbody weight than wild-type (+/+) and heterozygous mutant mice (−/+), asshown in FIG. 12. This difference in weight gain was present despite thefact that homozygous mutant mice consumed the same amount of foodcompared to wild-type and heterozygous mutant mice. When the GTT wasrepeated after high-fat diet challenge, it was again found thathomozygous mutant mice had decreased fasting glucose levels and improvedglucose tolerance when compared to wild-type and heterozygous controlmice. As shown in FIG. 13, significant differences in glucose levelswere seen between homozygous mutant mice (−/−) and wild-type controlmice (+/+) at all time points (p≦0.04). Significant differences werealso seen between homozygous mutant (−/−) and heterozygous mutant (−/+)mice at t=0 and t=30 minutes (p≦0.02).

A role for the glucagon receptor in diabetes and glucose tolerance isalso supported by the data presented in the previous examplesillustrating the effects of disruptions in glucagon receptors onpancreatic islet cells, body and organ weight, serum glucose levels, andbody fat percentage.

As is apparent to one of skill in the art, various modifications of theabove embodiments can be made without departing from the spirit andscope of this invention.

These modifications and variations are within the scope of thisinvention.

1. A transgenic mouse whose genome comprises a homozygous disruption inthe endogenous glucagon receptor gene, wherein the transgenic mouseexhibits, relative to a wild-type mouse, a pancreatic abnormality or ametabolic abnormality wherein the metabolic abnormality is selected fromthe group consisting of decreased fasting blood glucose level, increasedglucose tolerance, decreased fasting insulin level, increased glucagonlevel, decreased body weight, decreased body fat percentage, decreasedorgan weight, decreased body size and dwarfism.
 2. The transgenic mouseof claim 1, wherein the increased glucose tolerance is characterized bya decreased blood glucose level following glucose administration.
 3. Thetransgenic mouse of claim 1, wherein the pancreatic abnormality isselected from the group consisting of pancreatic hyperplasia, pancreatichypertrophy, increased cytoplasmic vacuolization of pancreatic cells,and increased cytoplasmic granularity of pancreatic cells.
 4. Thetransgenic mouse of claim 1, wherein the pancreatic abnormalitycomprises a pancreatic adenoma.
 5. The transgenic mouse of claim 1,wherein the pancreatic abnormality comprises an increase in number andsize of pancreatic alpha cells.
 6. The transgenic mouse of claim 1,wherein the pancreatic abnormality comprises a decrease in number ofpancreatic beta cells.
 7. A cell obtained from the transgenic mouse ofclaim 1.